CN104830714B - Red sage root endophyte with induction phenolic acid summation and application thereof - Google Patents
Red sage root endophyte with induction phenolic acid summation and application thereof Download PDFInfo
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- CN104830714B CN104830714B CN201510175259.4A CN201510175259A CN104830714B CN 104830714 B CN104830714 B CN 104830714B CN 201510175259 A CN201510175259 A CN 201510175259A CN 104830714 B CN104830714 B CN 104830714B
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Abstract
The present invention relates to a kind of natural medicinal plant endogenetic bacteria --- red sage root endophyte and application thereof.Specifically：It is Pseudomonas chlororaphis LG1 (Pseudomonas chlororaphis LG1), its preserving number is the invention discloses a kind of red sage root endophyte：CCTCC NO:M 2015082.The present invention further simultaneously discloses the purposes of the red sage root endophyte：Promote the accumulation of phenolic acid content in Hairy Root Cultures of Salvia miltiorrhiza.The present invention can solve the problem of active component content is low, quality is unstable in Hairy Root Cultures of Salvia miltiorrhiza incubation.
Description
Technical field
The present invention relates to a kind of natural medicinal plant endogenetic bacteria --- red sage root endophyte and application thereof.
Background technology
The red sage root (Salvia miltiorrhiza Bunge) is Labiatae, salvia, dicotyledonous, perennial straight
Vertical herbaceous plant, is a kind of traditional Chinese medicine of China, is used as medicine with root, stem.Red sage root use is quite varied, version in 2010《China
People's republic's pharmacopeia》Record, the red sage root can be used for treatment invigorate blood circulation, stasis-dispelling and pain-killing, relieving restlessness that clears away heart-fire cures mainly closed dysmenorrhea, the moon
Swollen and ache through uncomfortable, hot numbness, dysphoria and insomnia, angina pectoris etc..Modern medicine study finds that the red sage root is micro- in expansion coronary vasodilator, improvement
It is evident in efficacy in terms of circulation, it is used for the treatment of angiocardiopathy at present.With the continuous progress of biotechnology, to the red sage root
Pharmacological research is also more and more deep, and its market demand constantly expands.
The sales volume of compound danshen dripping pills is often only just more than 1,000,000,000 yuan by China according to statistics, annual red sage root Raw Material Demand
Amount reaches nearly 50,000 tons, and in rising trend year by year.The cultivation of the right red sage root and growth cycle length and active constituent content is low so that
Batch production and Clinical practice are extremely difficult；And it is wild in the case of the red sage root resource-constrained, excavation wild Salvia miltiorrhiza can destroy
The ecology of nature is steady.Therefore, using Plant Tissue Breeding or organ culture, effective ingredients in plant is directly obtained, will be into
For a kind of trend of development.Hairy root is because its hormone autotrophic, growth are rapid, the cycle is short, and completely metabolism is logical with host plant
The features such as road, and it is acknowledged as the raw material of preferably production Secondary metabolites.But hairy root also has secondary generation
Thank Product yields it is low as culture plant cell the problem of, these all affect the expansion culture or even industrialization of hairy root
Production.
Endophyte of plant is to separate or be obtained from the juice of inside plants from the plant tissue Jing Guo surface sterilization
, and the microorganism of obvious illness is not caused to the apparent upper non-hazardous of plant.Endophyte of plant includes benefit from each other
And neutral (neutral) endosymbiontic microorganism (mutualistic).
It is separated to endophyte from the various organs (root, stem, leaf, flower, fruit, seed) of many plants at present.It
Existing many document reports in terms of the generality in plant, abundant bio-diversity and functional diversity.Endophyte
Interacted by long-term with plant, more complicated relation is formed between them.Have now found that endophyte is planted to host
Thing plays the role of the following aspects：
(1) promote host plant growth, improve the productivity of host, host can be strengthened to disease, the resistance of worm, thus in agriculture
Important in inhibiting in terms of industry production, BIOLOGICAL CONTROL and biological control；
(2), resistance of the enhancing host plant to environment-stress.Such as to arid, global warning, saline and alkaline, heavy metal and
The resistance of other noxious material pollutions etc.；
(3) synthesis of host plant secondary metabolite, is promoted.The plant being grown in natural environment frequently suffer from it is bad because
Element such as microorganism invasion is coerced with injury, and in order to resist poor environment, plant cell often produces secondary metabolites.At present
It has been discovered that the accumulation of host plant secondary metabolite, such as Trichoderma can be promoted in a variety of endophytes
Atroviride, Streptomyces pactum etc..
In recent years, hairy root, the regulation and control of research Secondary Metabolism of Plant, promotion medicinal plant cometabolism are stimulated using elicitor
Product formation is always the focus studied both at home and abroad.The material that plant can be induced to produce secondary metabolites is called elicitor
(Elicitor), such as abiotic inducible factor of the biological inducible factor and metal ion etc. of yeast extract.Biological or non-
Under the induction of biotic factor, the resistance of plant can strengthen, while promoting the accumulation of botanical secondary metabolite.Microorganism is lured
Guide accesses hairy root, is the brand-new route for improving and obtaining Hairy Root Cultures of Salvia miltiorrhiza active constituent content, will fundamentally solve
Problems faced in the quality Control of the current red sage root and red sage root cultivation.
At present, the research both at home and abroad on the biological elicitor of the red sage root focuses primarily upon endogenetic fungus, but endogenetic bacteria is ground
Study carefully less, more promote medicinal plant secondary metabolite product without Pseudomonas chlororaphis (Pseudomonas chlororaphis)
Tired research report.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of red sage root endophyte --- and Pseudomonas chlororaphis, the bacterial strain can promote
Enter the accumulation of Hairy Root Cultures of Salvia miltiorrhiza phenolic acid, so as to solve that active component content in Hairy Root Cultures of Salvia miltiorrhiza incubation is low, quality
Unstable the problem of.
In order to solve the above-mentioned technical problem, the present invention provides a kind of red sage root endophyte, is Pseudomonas chlororaphis LG1
(Pseudomonas chlororaphis LG1), its preserving number is：CCTCC NO:M 2015082.
The purposes of sea of the invention simultaneously there is provided above-mentioned red sage root endophyte：Promote phenolic acid content in Hairy Root Cultures of Salvia miltiorrhiza
Accumulation.Phenolic acid is primarily referred to as Rosmarinic acid, tanshin polyphenolic acid B.
It is used as the improvement of the purposes of the red sage root endophyte of the present invention：Promote Hairy Root Cultures of Salvia miltiorrhiza weightening.
The red sage root endophyte is one plant of bacterium that inventor is separated to from red sage root plant, through conventional method identification and molecule
Identification shows that it belongs to Pseudomonas chlororaphis, is named as Pseudomonas chlororaphis LG1 (Pseudomonas chlororaphis
LG1)。
Separation method is：
1) after, the root of the red sage root is handled through surface sterilization, add sterilized water and be ground dilution, be coated on basic NA cultures
On base flat board, cultivated 1~3 day in 28~32 DEG C；
Basic NA medium slants formula：Peptone 10g, beef extract 3g, NaCl 5g, agar 18g and distilled water
1000mL, pH=7.2~7.4.
2), picking single bacterium falls within the purifying of identical culture medium (that is, basic NA culture mediums) plate streaking, in 28~32 DEG C of trainings
Support 1~3 day；
3), repeat step 2), until obtaining pure culture.
The preservation information of the red sage root endophyte of the present invention is specific as follows：Pseudomonas chlororaphis LG1Pseudomonas
Chlororaphis LG1, depositary institution：China typical culture collection center, preservation address：Wuhan, China Wuhan University, protects
Hide numbering：CCTCC NO:M 2015082, March 4 2015 preservation time.
Red sage root endophyte --- Pseudomonas chlororaphis LG1 (the hereinafter referred to as Pseudomonas chlororaphis CCTCC NO of the present invention:M
2015082) there is following biological property：
1. colony morphology characteristic：The well-grown on NA culture mediums, bacterial community feature：Bacterium colony is rounded, crocus, light
Sliding, neat in edge, bacterium colony central protuberance produces orange pigment.
2. morphological features：Rod-short, Gram-negative.
3. major physiological biochemical character：The bacterium can on NA culture mediums, growth temperature be 20~40 DEG C, pH value be 6.0
Well-grown in~8.0 environment.
4. genetics characteristics：According to《Common bacteria system identification handbook》(east show pearl, Cai Miaoying, Science Press,
2001), Pseudomonas chlororaphis CCTCC NO of the invention:In the morphological features of M 2015082, the false unit cell of green pin with country's name
Bacterium (Pseudomonas chlororaphis) is closest；Pseudomonas chlororaphis CCTCC NO:M 2015082 16S rDNA
Sequence analysis shows, with the strain Pseudomonas chlororaphis strain recorded in Genbank international data centers
KY5406 (KM030060.1) similitude is 99%.Therefore, Pseudomonas chlororaphis CCTCC NO:M 2015082 should belong to green
Pin pseudomonad.
The Pseudomonas chlororaphis CCTCC NO of the present invention:M 2015082 can promote phenolic acid in Hairy Root Cultures of Salvia miltiorrhiza to contain
The accumulation of amount, can promote hairy root fresh weight to increase by 11.3%, rosmarinic acid contents improve 111%, and content of danshinolic acid B is improved
72%.It is nontoxic to people and animals, environment is not polluted.It can be applied, be produced necessarily in Hairy Root Cultures of Salvia miltiorrhiza culture
Economic benefit.
When the red sage root endogenetic bacteria of the present invention is used to promote the accumulation of Hairy Root Cultures of Salvia miltiorrhiza phenolic acid, only need that the pellet will be utilized
Join endogenetic bacteria --- Pseudomonas chlororaphis CCTCC NO:Elicitor prepared by M 2015082 is connected in hairy root culture medium, i.e.,
The effect for improving Hairy Root Cultures of Salvia miltiorrhiza phenolic acid content can be obtained.
Embodiment
Embodiment 1, Pseudomonas chlororaphis CCTCC NO:M 2015082 isolated culture method, carries out following walk successively
Suddenly：
1st, the healthy red sage root plant of plantation is gathered in Sichuan Zhong Jiang, its root is fully cleaned with running water, 25KHz ultrasounds
Ripple is handled 5 minutes；
2nd, aseptically, to the aseptic water washing 2 times of the material to be tested obtained by step 1, then successively with 75% (v/
V) alcohol and 3% (m/V) liquor natrii hypochloritis carries out surface sterilization to material to be tested, so as to kill the micro- life in material to be tested surface
Thing；
3rd, in gnotobasis, by the aseptic water washing 3 times of the material to be tested obtained by step 2, last 1 punching of 100 μ l is taken
Aseptic water washing liquid coating produced by washing is inoculated on basic NA culture mediums, and after 30 DEG C of culture 24h, observation whether there is bacterium colony production
It is raw.Verify whether above-mentioned sterilization method all kills material to be tested surface microorganism accordingly.
If observation conclusion is：There is no bacterium colony generation, then carry out following steps 4；If observation conclusion is：There is bacterium colony production
Give birth to, then return to step 2.
4th, in super-clean bench, the above-mentioned red sage root samples handled well of 1~2g is taken, are fully ground in sterile mortar, added
5mL sterilized waters, are mixed, and are stood 15min, are taken 100 μ l supernatants dilution spreads in basic NA solid mediums, be placed in 28~32 DEG C
(preferable 30 DEG C) are cultivated 1~3 day；
NA solid mediums：Peptone 10g, beef extract 3g, NaCl 5g, agar 18g and distilled water 1000mL, pH=7.2
~7.4.
5th, the strain grown on picking NA solid mediums is placed in 28~32 DEG C in same medium line purifies and separates
(preferable 30 DEG C) are cultivated 1~3 day, picking single bacterium colony；
Aforesaid operations are repeated untill single bacterium colony is obtained.
6th, single bacterium colony resulting in step 5 is inoculated in into basic NA medium slants to be stored in 4 DEG C of refrigerators.
Obtain Pseudomonas chlororaphis CCTCC NO:M 2015082.
The preparation method of embodiment 2, the elicitors of Pseudomonas chlororaphis CCTCC M 2015082, is followed the steps below successively：
1st, by Pseudomonas chlororaphis CCTCC NO:M 2015082 is transferred to basic NA medium slants, and 30 DEG C are cultivated 2 days；
Obtain inclined-plane seed；
Basic NA medium slants formula：Peptone 10g, beef extract 3g, NaCl 5g, agar 18g and distilled water
1000mL, pH=7.2~7.4.
2nd, with the slant strains obtained by the ring step 1 of oese picking one, it is inoculated in equipped with 100ml NA fluid nutrient mediums
In 250ml triangular flasks, in 28 DEG C with 220r/m speed oscillation culture 72h, obtain Pseudomonas chlororaphis CCTCC M 2015082 and ferment
Stoste；
NA Liquid Culture based formulas is：Peptone 10g, beef extract 3g, NaCl 5g and distilled water 1000mL, pH=7.2~
7.4。
Above-mentioned steps 1 and step 2 are cultivated under the conditions of natural light.
3rd, step 2 gained fermenation raw liquid is taken, in 4 DEG C, 12000rpm centrifuges 2min, takes supernatant to cross 0.22 μm of miillpore filter,
Gained filtrate is the elicitors of Pseudomonas chlororaphis CCTCC M 2015082.
The promotion that embodiment 3, Pseudomonas chlororaphis CCTCC M 2015082 are accumulated to Hairy Root Cultures of Salvia miltiorrhiza phenolic acid is real
Test：
1st, 0.3g Hairy Root Cultures of Salvia miltiorrhiza (female root) is kept away in 100ml 6,7-V culture mediums under conditions of 25 DEG C, 110rpm
Optical culture 18 days；Based on thing；
Experimental group：1.5ml Pseudomonas chlororaphis CCTCC elicitors (gained of embodiment 2) are added in each base,
Continue the same terms (lucifuge, 25 DEG C, 110rpm) to cultivate 6 days；It is used as experimental group；
Blank control group：1.5ml sterile NA fluid nutrient mediums are added in each base, continues the same terms and (keeps away
Light, 25 DEG C, 110rpm) cultivate 6 days；It is used as blank control group；
Above-mentioned experimental group and blank control group set 5 repetitions respectively；
Hairy Root Cultures of Salvia miltiorrhiza obtained by experimental group and blank control is proceeded as follows respectively：
2nd, the hairy root that step 1 is obtained is cleaned three times with distilled water, fully sucked with filter paper after moisture, claim fresh weight
(fresh weight, FW), calculates its increased times (increased times=(increment-inoculum concentration)/inoculum concentration)；With will claim
The hairy root of fresh weight is put into baking oven, and 55 DEG C of dryings weigh dry weight (dry weight, DW) to constant weight.
3rd, the red sage root dried to constant weight in right amount, grind into powder are taken.0.5g is taken to add 2.5mL 70% (volume %) first
Ultrasonic extraction 60min in alcohol, 120W supersonic wave cleaning machines (water temperature can not be too high in ultrasonic procedure, can be suitably added ice cube, i.e.
Control temperature≤20 DEG C), supernatant is taken after cooling, 0.45 μm of miillpore filter is crossed, is carried out with the method for high performance liquid chromatography (HPLC)
The measure of phenolic acid content.
Measurement result is as follows：
The hairy root fresh weight average specific blank pair of the elicitors of Pseudomonas chlororaphis CCTCC M 2015082 is added in experimental group
11.3% is added according to group, rosmarinic acid contents are higher than blank control group by 111%, and content of danshinolic acid B is higher than blank control group
72%.It is specific as described in Table 1.
Comparative example, the Pseudomonas chlororaphis CCTCC M selected respectively in following Pseudomonas chlororaphis alternate embodiment 2
2015082 carry out elicitor preparation, and the elicitor of gained is detected that acquired results are as described in Table 1 according to embodiment 3.
The Pseudomonas chlororaphis that A, the Code Number of Germany Microbiological Culture Collection Center DSMZ preservations are DSM 12972；
The green pin that B, the Code Number of Sichuan Province's microbial collection center preservation Culture Collection are SCTCC 100003
Pseudomonad；
The green pin that C, the Code Number of Sichuan Province's microbial collection center preservation Culture Collection are SCTCC 100343
Pseudomonad；
D, the Code Number of Chinese agriculture Microbiological Culture Collection administrative center preservation Culture Collection are ACCC's 05437
Pseudomonas chlororaphis；
The influence that the Pseudomonas chlororaphis CCTCC M 2015082 of table 1. are accumulated to Hairy Root Cultures of Salvia miltiorrhiza phenolic acid
It is laboratory mean values ± standard deviation in table, * * indicate pole significant difference, P<0.01, * represents that there were significant differences,
P<0.05
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (3)
1. red sage root endophyte, it is characterized in that：For Pseudomonas chlororaphis LG1 (Pseudomonas chlororaphis LG1), its
Preserving number is：CCTCC NO:M 2015082.
2. the purposes of red sage root endophyte as claimed in claim 1, it is characterized in that：Phenolic acid in Hairy Root Cultures of Salvia miltiorrhiza is promoted to contain
The accumulation of amount, the phenolic acid is Rosmarinic acid, tanshin polyphenolic acid B.
3. the purposes of red sage root endophyte according to claim 2, it is characterized in that：Promote Hairy Root Cultures of Salvia miltiorrhiza weightening.
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| CN201510175259.4A CN104830714B (en) | 2015-04-15 | 2015-04-15 | Red sage root endophyte with induction phenolic acid summation and application thereof |
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| CN201510175259.4A CN104830714B (en) | 2015-04-15 | 2015-04-15 | Red sage root endophyte with induction phenolic acid summation and application thereof |
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| Publication Number | Publication Date |
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| CN104830714B true CN104830714B (en) | 2017-11-07 |
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| 一株促进丹参生长和提高丹酚酸含量的活性内生真菌;唐坤等;《菌物学报》;20140515;第33卷(第3期);第594-600页 * |
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