CN106434406B - It is a kind of generate borrelidin actinomyces and its application - Google Patents

It is a kind of generate borrelidin actinomyces and its application Download PDF

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CN106434406B
CN106434406B CN201610229401.3A CN201610229401A CN106434406B CN 106434406 B CN106434406 B CN 106434406B CN 201610229401 A CN201610229401 A CN 201610229401A CN 106434406 B CN106434406 B CN 106434406B
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borrelidin
actinomyces
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methanol
fermentation liquid
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CN106434406A (en
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万传星
罗晓霞
周彪
胡文超
周忠波
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Tarim University
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons

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Abstract

The invention discloses a kind of superior strain (yield reach at 10-15mg/L) for generating borrelidin, be Ghana streptomycete (S.ghanaensis) TRM78-24, on November 2nd, 2015 in China typical culture collection center, deposit number is CCTCC NO:M201565.Application the invention also discloses the borrelidin of method and the actinomyces and its generation using bacterial strain production borrelidin in terms of preventing and treating cotton verticillium wilt simultaneously.

Description

It is a kind of generate borrelidin actinomyces and its application
Technical field
The present invention relates to agricultural and field of medicaments, and in particular to one kind can generate the actinomyces new sources of borrelidin Bacterial strain and its method using bacterial strain production borrelidin and its application in antagonism cotton verticillium wilt.
Background technique
Borrelidin (also known as Boli mycin, Borrelidin) is the cyanogen two for having cyclopentane-carboxylic acid and conjugation 18 membered macrolide compounds of alkene feature structure, earliest by Berger et al. in 1949 from Lou Che Shi streptomycete It is isolated in (Streptomycete rochei) metabolite.Borrelidin is in addition to extensive antibacterium, resisting very Other than bacterium and Antimalarial, also there are good inhibition new vessels to generate activity (IC50=0.8nM), it is current anti-tumor drug One of hot spot compound of research and development.Patent (CN1732264A) discloses the polyketide synthase for generating borrelidin And application thereof；Preventive and therapeutic effect of the patent (CN103442564B) to soybean phytophthora root rot.The invention discloses one plant of productions to dredge The superior strain of spiral voxel and its method for producing borrelidin using the bacterial strain, and utilize the bacterial strain and the compound Application in prevention and treatment cotton verticillium wilt evil.
Summary of the invention
The present invention provides a kind of actinomyces for generating borrelidin, and the actinomyces have cotton spoting verticillium wilt good Control efficiency is Ghana streptomycete (Streptomycesghanaensis) TRM78-24, on November 2nd, 2015 is in Chinese allusion quotation (preservation address is: Wuhan City, Hubei Province Wuhan University, postcode: 430072), deposit number is the preservation of type culture collection CCTCC NO:M2015656, through detecting, result is survival.
The actinomyces are generating the application in borrelidin.The application, it is characterised in that including following steps It is rapid: to cultivate the actinomyces under suitable conditions, obtain fermentation liquid；Borrelidin is isolated and purified from fermentation liquid.
Preferably, the culture medium prescription and condition of culture of the actinomyces are cultivated are as follows: millet 10g/L, glucose 10g/L , peptone 3g/L, CaCO3 2g/L, NaCl 2.5 g/L, adjusting pH are 7.2-7.4；Fermentation training is inoculated in by 8% inoculum concentration It supports in base, 28 DEG C, shaking table culture 7 days under the conditions of 180rpm.
Preferably, the method for borrelidin is isolated and purified from fermentation liquid specifically: after fermentation liquid filtration, wet process loading To D101 or AB-8 macroporous resin chromatography；Then it is eluted respectively with 50% ethanol solution, 95% ethanol solution；95% ethanol elution Liquid is concentrated under reduced pressure after removing solvent and obtains the medicinal extract rich in borrelidin；Then by compression leg chromatographic isolation in ODS, successively It is eluted with 50% methanol-water and 100% methanol.100% methanol elution position then prepares borrelidin with 70% methanol-water Sterling.
The invention further relates to the actinomyces in the application for preventing and treating cotton verticillium wilt.Preferably, the unwrapping wire is used The fermentation liquid of bacterium, or its Active components is further separated for preventing and treating cotton verticillium wilt.
The invention has the following advantages: Ghana streptomycete (S.ghanaensis) TRM78-24 main metabolites For borrelidin, the content that HPLC analyzes borrelidin in how wholesale zymotic fluid is 10-15mg/L, borrelidin and its Producing strains Ghana streptomycete (S.ghanaensis) fermentation liquid of TRM78-24 has good control efficiency to cotton spoting verticillium wilt.
Detailed description of the invention
The electromicroscopic photograph (showing fibrillae of spores and spore shape) of Fig. 1 Ghana streptomycete TRM78-24；
The chemical structure of Fig. 2 borrelidin；
Fig. 3 borrelidin1HNMR spectrogram；
Fig. 4 borrelidin13CNMR spectrogram；
The hsqc spectrum figure of Fig. 5 borrelidin；
The HMBC spectrogram of Fig. 6 borrelidin；
The NOESY spectrogram of Fig. 7 borrelidin；
Fig. 8 Ghana streptomycete (S.ghanaensis) TRM78-24 16S rDNA sequence.
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not to limit of the invention System, only illustrates.
One Ghana streptomycete of embodiment (S.ghanaensis) TRM78-24 source be separately cultured
From the 0-30cm topsoil sample of Xinjiang Alar Area cotton field, using dilution spread flat band method, with Gao Shi mono- Number culture medium, 28 DEG C of constant temperature incubations, separation obtain bacterial strain TRM78-24, which shows to cotton spoting verticillium wilt bacterium Antagonistic activity.
Two Ghana streptomycete of embodiment (S.ghanaensis) TRM78-24 identification
(1) Ghana streptomycete (S.ghanaensis) TRM78-24 Microscopic Identification
For the electromicroscopic photograph of Ghana streptomycete TRM78-24 referring to Fig. 1, Electronic Speculum shows that its spore is ellipsoid, fibrillae of spores spiral Zhuan You branch.
(2) Ghana streptomycete (S.ghanaensis) TRM78-24 molecular biology identification
1 design of primers
Its sequence of the actinomyces universal primer of use is respectively as follows: 27F(5 '-AGAGTTTGATCCTGGCTC-3 ') and 1492R (5 '-CGGCTACCTTGTTACGACTT-3 '), by AudioCodes biotechnology (Beijing), Co., Ltd is synthesized.
The extraction of 2 Biocontrol Strain TRM78-24 genomic DNAs
The TRM78-24 thallus collected on culture plate is put into the sterile centrifugation tube of 1.5 mL, and 1 × TE of 480 μ L is added Buffer.20 μ L lysozyme (50 mgmL are added-1), it is put into 37 DEG C of shaking tables, 200rpm shaken overnight.50 μ L are added in every pipe 5 μ L, 20 mgmL is added in 20% SDS-1Proteinase K, be put into 60 DEG C of shaking tables, 200 r/min vibrate 1 h.550 μ are added The phenol of L: chloroform: isoamyl alcohol (25:24:1), 12000rpm are centrifuged 10 min, take supernatant to move into another centrifuge tube, extract repeatedly 2-3 times.Supernatant is taken, isometric dehydrated alcohol is added, the sodium acetate (3molL of 0.1 times of volume is added-1), it is put into 4 DEG C of refrigerators Middle precipitating DNA about 0.5h.12000rpm is centrifuged 10min, abandons supernatant.Centrifugation product 2 times is cleaned with 70% ethyl alcohol of 200 μ L, 12000rpm is centrifuged 5 min, abandons supernatant, ethyl alcohol is volatilized complete.Sufficiently dissolve the DNA of bottom with the 50 sterile ultrapure waters of μ L, 1% Agarose gel electrophoresis detection DNA extract quality, the DNA of extraction is put into -20 DEG C of refrigerators and is saved backup.
The amplification of 3 Biocontrol Strain TRM78-24 16S rDNA genes
With actinomyces 16S rDNA gene universal primer 27F(5 '-AGAGTTTGATCCTGGCTC-3 ') and 1492R(5 '- CGGCTACCTTGTTACGACTT-3 ') amplification actinomyces genomic DNA in 16S rDNA genetic fragment.
The PCR reaction system of 25 μ L are as follows: ddH220.4 μ L, 10 × Buffer(buffer of O contains Mg2+) 2.5 μ L, dNTPs 0.5 μ L, 7F(10 μm of olL of primer 2-1) 0.5 μ L, 1492R(10 μm of olL of primer-1) 0.5 μ L, Taq archaeal dna polymerase, 0.1 μ L, 0.5 μ L of template DNA.
PCR reaction condition are as follows: 94 DEG C of 4 min of initial denaturation；94 DEG C of 1min are denaturalized, anneal 56 DEG C of 1 min, extends 72 DEG C 2 min, 30 circulations；72 DEG C of 8min of overall elongation.It is detected after the reaction was completed with 1% agarose gel electrophoresis.Qualified PCR Product carries out sequencing.
4. the 16S rDNA sequencing results of biocontrol microorganisms TRM78-24
Sequencing result DNAMAN5.2 software splices, and determines that the segment by 1461 base compositions, obtains TRM78-24 16S rDNA sequence refer to Fig. 8.
It is compared by GenBank database http://www.ezbiocloud.net/ BLAST, biocontrol microorganisms TRM78-24 With Ghana streptomyceteStreptomyces ghanaensisThe 16S rDNA gene order of KCTC 9882 (T) is most like (consistency 99.93%), thus by the biocontrol microorganisms be accredited as Ghana streptomycete (S.ghanaensis) TRM78-24.
The utilization of embodiment three Ghana streptomycete (S.ghanaensis) TRM78-24 ferments and prepare borrelidin
Inoculation Ghana streptomycete (S.ghanaensis) a small amount of conidia powder of TRM78-24 is in ISP4 culture medium, 28 DEG C of cultures 4d obtains seed liquor, takes the inoculum concentration of 2mL(8 %) seed liquor is inoculated in fermented and cultured in millet fermentation medium, fermentation condition Are as follows: revolving speed 180 rpm, initial pH 7.2-7.4, liquid amount 250mL, 28 DEG C of fermentation temperature, ferment 7d.Fermentation liquid is collected, with four Layer filtered through gauze fermentation liquid, wet process are loaded to macroporous resin chromatography (the every 100L filling D101 or AB-8 macropore tree of ferment filtrate Rouge 2.0kg)；Then it is eluted respectively with 50% ethanol-water solution, 95% ethanol-water solution；95% ethanol eluate is concentrated under reduced pressure 95% ethanol extract rich in borrelidin is obtained after removing solvent, then by compression leg chromatography in ODS, respectively with 50% first Alcohol-water and the elution of 100% methanol.100% methanol elution position then prepares borrelidin sterling with 70% methanol-water, For HPLC analysis shows that the content of borrelidin is 12.0mg/L in the fermentation liquid, practical preparation amount is 6.6mg/L.
The Structural Identification of example IV borrelidin
From Ghana streptomycete (S.ghanaensis) compound that isolates and purifies in TRM78-24 fermentation liquid is greyish white toner End； 1H H NMR spectroscopy show 3 alkene hydrogen signals (δ H6.28,6.57,6.87), 4 methyl signals (δ H 0.83, 0.84, 0.85,1.02) (Fig. 3 is referred to), 13C H NMR spectroscopy show there are two carbonyl (δ C173.2,180.5), 4 olefinic carbon signals (δ C 140.2,129.0,145.4,119.9), 3 company's oxygen carbon signals (δ C77.4,73.1,72.9) (referring to Fig. 4).Pass through ratio To document (The Journal of Antibiotics 42 (6): 1006-1007) and according to hsqc spectrum (referring to Fig. 5), It is borrelidin (borrelidin) (Fig. 2) by the compound identification, NMR signal belongs to table 1.The long-range correlativity of HMBC Further confirm reliability of structure (referring to Fig. 6,7).
1 borrelidin of table1H NMR and13C NMR spectral data
Table 11H NMR and 13C NMR Data for borrelidin (600 and 200 MHz in MeOD) a
a All assignments are based on HSQC and HMBC experiments.
Antibacterial activity of five borrelidin of embodiment to cotton spoting verticillium wilt
Cultured cotton verticillium wilt disease fungus spore is eluted from culture plate with sterile water, is made 106-107It is a Spore/mL spore suspension, on the spore suspension to PDA plate for drawing 100 μ L, with sterilized spreading rod by bacteria suspension Coating uniformly, slightly dries up on superclean bench.It is equidistantly placed Oxford cup on plate carrying disease germs, is added in each Oxford cup The borrelidin of 200 μ L various concentration gradients is control, 28 DEG C of culture 6-7d, crossing method measurement suppression with equivalent acetone Bacterium loop diameter, every processing are repeated 3 times.The result shows that borrelidin inhibits verticillium dahliae spore germination and mycelia growth EC50For 60 μ g/mL.

Claims (8)

1. a kind of actinomyces for generating borrelidin, which is characterized in that it is Ghana streptomycete (Streptomycesghanaensis) TRM78-24, on November 2nd, 2015 is in China typical culture collection center preservation, deposit number CCTCC NO:M201565.
2. application of the actinomyces as described in claim 1 in production borrelidin.
3. application as claimed in claim 2, which is characterized in that include the following steps: to cultivate the actinomyces, be fermented Liquid；Borrelidin is isolated and purified from fermentation liquid.
4. application as claimed in claim 3, which is characterized in that cultivate the culture medium prescription and condition of culture of the actinomyces Are as follows: millet 10g/L, glucose 10g/L, peptone 3g/L, CaCO3 2g/L, NaCl 2.5 g/L, adjusting pH are 7.2-7.4； It is inoculated in fermentation medium by 8% inoculum concentration, 28 DEG C, cultivate under the conditions of 180rpm.
5. application as claimed in claim 4, which is characterized in that incubation time is 5-8 days.
6. such as the described in any item applications of claim 3 to 5, it is characterised in that: isolate and purify borrelidin from fermentation liquid Method specifically:
After fermentation liquid filtration, wet process is loaded to D101 or AB-8 macroporous resin chromatography；Then respectively with 50% ethanol solution, 95% Ethanol solution elution；95% ethanol eluate is concentrated under reduced pressure after removing solvent and obtains the medicinal extract rich in borrelidin；Then lead to Compression leg chromatographic isolation in ODS is crossed, is successively eluted with 50% methanol-water and 100% methanol；Then 70% is used in 100% methanol elution position Methanol-water prepares borrelidin sterling.
7. actinomyces as described in claim 1 are in the application of prevention and treatment cotton verticillium wilt.
8. the use as claimed in claim 7, it is characterised in that: it uses the fermentation liquid of the actinomyces, or further separation Its Active components is for preventing and treating cotton verticillium wilt.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1732264A (en) * 2002-12-27 2006-02-08 百奥帝卡技术有限公司 Borrelidin-producing polyketide synthase and its use
CN103409341A (en) * 2013-07-08 2013-11-27 浙江工业大学 Application of relA gene in increase of moenomycin yield of streptomyces bambergiensis and strain
CN103442564A (en) * 2011-03-25 2013-12-11 浙江海正药业股份有限公司 Effect of borrelidin for controlling soybean phytophthora root rot

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1732264A (en) * 2002-12-27 2006-02-08 百奥帝卡技术有限公司 Borrelidin-producing polyketide synthase and its use
CN103442564A (en) * 2011-03-25 2013-12-11 浙江海正药业股份有限公司 Effect of borrelidin for controlling soybean phytophthora root rot
CN103409341A (en) * 2013-07-08 2013-11-27 浙江工业大学 Application of relA gene in increase of moenomycin yield of streptomyces bambergiensis and strain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
一株加纳链霉菌种子培养工艺及移种标准的研究;黄艳等;《中国畜牧杂志》;20140410;第50卷(第7期);第79-84页 *

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