CN107034145B - Desmopsis virescens CAMT66351 and application thereof - Google Patents
Desmopsis virescens CAMT66351 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a pestalotiopsis fungus and application thereof, namely, separation and character change analysis of cordyceps sinensis endophytic fungi are carried out, the body and the paw of mole cricket are cultured, separated and purified by using different culture mediums, and the pestalotiopsis fungus is separated and screened out, and is preserved in Guangdong province microorganism strain preservation center in 2016, 12, 26 days, the preservation number is GDMCC No: 60135. the fungus has the best growth condition under the condition of rice culture medium, 20 ℃ and no light, can produce nucleoside with higher content, is expected to realize the massive fermentation and production of nucleoside in vitro by finding the fungus, and has wide application value.
Description
Technical Field
The invention relates to the technical field of microorganisms, and in particular relates to a Desmopsis virginiana CAMT66351 and application thereof.
Background
The mole cricket worm grass is a thallus polypide complex with a growth mode similar to that of cordyceps sinensis, namely, fungus infects mole cricket polypide, decomposes tissue in the polypide and utilizes nutrient substances of the mole cricket polypide to obtain an energy source, and then the mole cricket worm grass is formed. Although the mole cricket cordyceps sinensis is rare, the effective active substances contained in the cordyceps sinensis have the medical and health care efficacy. Wild cordyceps sinensis grows in high-altitude areas, and the cordyceps sinensis contains 15 trace elements such as amino acids, nucleosides, sugars, alcohols, vitamins and organic acids. The Chinese herbal medicine gang mu Shi Yi (supplement to compendium of materia medica) mentions that the cordyceps has the same function as ginseng, and in addition, the pharmacological functions of the cordyceps comprise the treatment of asthma, bronchus and inflammation, the treatment of the kidney, the treatment of nephritis, the immune regulation and the treatment of the old and general weakness after illness. In addition, the inventor finds that the cordyceps sinensis extract can reduce lymphadenopathy, relieve proteinuria and improve renal function by establishing an animal model. Therefore, the cordyceps is always regarded as a precious tonic and is called as 'three treasures of traditional Chinese medicine' together with the pilose antler and the ginseng. However, with the destruction of ecological environment in recent years, the cordyceps sinensis resource is more and more poor, the price is increased all the way, and the cordyceps sinensis resource is expanded from six jin and seven hundred yuan in the middle of the nineties of the last century to sixteen seventy thousand at present. Fortunately, people find that a large number of strain without characters is obtained by artificial planting, active substances in the strain are extracted after independent culture, and the strain can become a novel product when being applied to the food and medicine industry.
The cordyceps sinensis endophytic strains are various in types, symbiotic, concomitant or xenobiotic with cordyceps sinensis, and are separated to generate metabolites, natural cordyceps sinensis and a microenvironment thereof contain various endophytic bacteria, and the nutritional value of some endophytic bacteria is even higher than that of the cordyceps sinensis, so that the cordyceps sinensis is a great direction for research, and a new cordyceps sinensis variety with similar nutritional value to the cordyceps sinensis and cordyceps militaris is developed. Therefore, the morphological study of the cordyceps strain is extremely important. At present, there are many reports on cordyceps sinensis, but there are few reports on mole cricket cordyceps sinensis, particularly on the isolation of asexual strains therefrom.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides the wassermei pestalotiopsis CAMT66351 separated from the mole cricket cordyceps sinensis.
The second purpose of the invention is to provide the application of the Desmopsis virescens CAMT 66351.
The purpose of the invention is realized by the following technical scheme:
a Pestalotiopsis victorialis CAMT66351 (Latin article is Pestalotiopsis virsiaeCAMT 66351), which is preserved in Guangdong province collection of microorganisms and strains in 2016, 12 and 26 days, and the preservation number is GDMCC No: 60135.
preferably, the fungus colony grows irregularly, has a dry surface and is milky white with spores scattered; with hypha, no septum, and granular spore.
Preferably, the fungal ITS sequence is as set forth in SEQ ID NO: 1 is shown.
The invention also provides a microbial preparation containing the Desmopsis virescens CAMT 66351.
The invention also provides application of the Desmopsis virescens CAMT66351 in nucleoside production; preferably, the nucleosides are adenosine, guananthine, uridine and inosine.
The invention also provides a method for producing nucleoside, which is implemented by inoculating the pestalotiopsis virginiana CAMT66351 in a liquid culture medium and culturing for a period of time at 20 ℃.
More preferably, the pestalotiopsis virescens CAMT66351 is inoculated in a rice liquid culture medium and cultured for about 7 days at 20 ℃ under the condition of no illumination.
Specifically, the liquid culture medium is a rice culture medium, and the rice culture medium comprises the following components: 45g of rice, 20g of glucose, 10g of peptone, 10g of yeast extract powder and KH2PO42g、MgSO42g of water are added until 1000m L is reached, and the pH is adjusted to 5.5.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for separating and analyzing character change of cordyceps sinensis endophytic fungi, which comprises the steps of culturing, separating and purifying mole cricket bodies and paws by using different culture media, separating and screening a wassme pleiotropus vesidoides CAMT66351, wherein the fungi are preserved in Guangdong province microorganism culture collection center in 2016, 12 and 26 days, and the preservation numbers are GDMCC No: 60135. the fungus has the best growth condition under the condition of rice culture medium, 20 ℃ and no light, can produce nucleoside with higher content, is expected to realize the massive fermentation and production of nucleoside in vitro by finding the fungus, and has wide application value.
Drawings
FIG. 1 is an HP L C profile of the standard.
FIG. 2 is an HP L C map of CAMT 66351.
FIG. 3 is a developmental tree of CAMT 66351.
FIG. 4 is a microscopic image (400 times) of CAMT 66351.
FIG. 5 is a colony morphology map of CAMT 66351.
Detailed Description
The invention will be further described with reference to the drawings and specific examples, which are not to be construed as limiting the invention; modifications and substitutions of the methods, procedures, and conditions of the present invention can be made without departing from the spirit and substance of the invention. Unless otherwise indicated, the experimental procedures used in the examples are all conventional procedures and techniques well known to those skilled in the art, and reagents or materials are all commercially available.
Example 1 Strain isolation, screening and identification
Firstly, sample treatment
The mole cricket cordyceps sinensis sample is completely packaged by a nylon-polyethylene bag (7 × 10cm transparent food vacuum bag) and stored at low temperature for standby, the mole cricket cordyceps sinensis sample is washed by tap water for 15min, then washed by sterile normal saline for 3 times, then disinfected by 75% ethanol, finally washed by sterile normal saline, then the worm body and the paws are separated and cut into pieces by tweezers and scissors which are dried and sterilized at high temperature on a sterile operating table, 5.0g of each worm body sample and each paws sample are weighed and respectively placed in a conical bottle containing 45m L normal saline, and 1: 10 sample uniform liquid, namely worm body sample liquid and paws sample liquid, is prepared.
Preparing a sterile test tube containing 9m L of physiological saline, sucking 1: 10 of sample liquid 1m L by using a 1m L sterile pipette, slowly injecting the sample liquid along the wall of the test tube, fully shaking the test tube to uniformly mix the sample liquid and the physiological saline to prepare 1: 100 of sample uniform liquid, and preparing 1: 1000 of sample uniform liquid according to the same method.
Insect body sample liquid: respectively diluting to 10-1、10-2、10-3Three gradients; sealing and placing in a refrigerator for standby.
Claw sample liquid: respectively diluting to 10-1、10-2、10-3Three gradients; sealing and placing in a refrigerator for standby.
Second, culture medium formula
(1) Rice culture medium: weighing rice 45g, soaking in distilled water for softening, grinding in mortar to obtain small granules or completely grinding, filtering with gauze to obtain turbid rice water, adding glucose 20g, peptone 10g, yeast extract powder 10g, and KH2PO42g、MgSO42g, adding 15g of agar powder into the solid culture medium, adding water to 1000m L, and adjusting the pH value to 5.5.
(2) Potato culture medium: peeling potatoes, weighing 45g of peeled potatoes, boiling until the potatoes are completely softened, and filtering the potatoes with gauze to remove residues to obtain filtrate; adding glucose 20g, peptone 10g, yeast extract 10g, KH2PO42g、MgSO42g, 15g of agar powder is added into the solid medium, water is added to 1000m L at the same time, and the pH value is adjusted to 5.5.
(3) PDA culture medium: prepared according to the medium instruction.
Boiling the three culture mediums for several minutes, cooling to 50 deg.C, subpackaging in triangular flasks, wrapping with kraft paper, placing into a vertical pressure steam sterilizer, adjusting to 121 deg.C, sterilizing for 20min, cooling to 50 deg.C, and pouring into flat plate.
Thirdly, strain separation and culture
(1) The plate coating method comprises cooling sterilized culture medium to 50 deg.C, pouring into sterile plate, cooling, solidifying, adding 1m L sterile pipette, respectively, dropwise adding the gradient diluent 1m L onto the surface of the plate, and uniformly coating with sterile coating rod.
(2) The dilution plate method comprises respectively taking 1m L of each gradient of the dilution solution with a 1m L sterile pipette, dripping onto a plate, pouring the culture medium cooled to 50 deg.C into the sterile plate, and slowly rotating to mix the sample solution with the culture medium.
(3) A flat plate scribing method: pouring the culture medium with the temperature of 50 ℃ into a sterile plate to prepare a sterile plate, cooling and solidifying, dipping the dilution liquid with each gradient by using an inoculating loop, and continuously scribing on the sterile plate; and (5) after the bacterial liquid on the surface of the flat plate is absorbed, turning over the flat plate, culturing in a constant-temperature incubator at 20 ℃, and observing and recording the growth condition of the bacterial colony.
The experimental result shows that the shapes of the colonies shown by the potato culture medium and the rice culture medium are not obviously different, but are obviously different from the colonies in the PDA culture medium, and the colonies are mainly shown in small number and even do not grow. Wherein the colony number of the rice culture medium is 12, the colony comprises colonies obtained by separating paws and polypide, the potato culture medium is 9, and the PDA culture medium is 1.
Fourthly, strain purification and subculture
Colonies having morphological characteristics of typical cordyceps are selected from the culture medium and subjected to purification culture, i.e., changes in colony morphology are observed according to changes in culture time under the same culture conditions. The colony morphology is stable after primary separation, second generation purification, third generation purification, fourth generation purification and fifth generation purification.
Counting the number of colonies, and selecting the most representative colonies for subculture. There are 12 bacterial colonies primarily separated from the worm sample liquid and the paw sample liquid. The colony obtained by preliminary separation is disordered and has 12 kinds, which are respectively named as No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10 and No. 11, the colony obtained by preliminary separation is purified and cultured for the first generation, and the colony obtained by microscopic observation after 7 days is obtained, so that the colony obtained by purification for the second generation has 2 kinds, wherein the colony is the same kind of the No. 4, the No. 5 and the No. 6, and the colony has the same kind of the No. 7, the No. 8, the No. 9, the No. 10 and the No. 11. Culturing to the third generation, and culturing for the fourth and fifth generations to find that the colony morphology has no obvious change and tends to a stable state, and the change of the colony morphology is shown in table 1.
TABLE 1 colony morphology change map
Fifth, microscopic morphology observation of colonies
Selecting strains with good growth for microscopic observation, performing on an aseptic operating platform, burning red and sterilizing an inoculating loop under an alcohol lamp, selecting bacterial colonies with good growth from a solid culture medium, lightly picking or scraping hyphae, placing on a glass slide on which a staining solution is dropped, gently spreading the hyphae, wherein the staining solution is gossypol blue, crystal violet and 75% alcohol respectively, absorbing excessive moisture by using filter paper, covering a cover glass, and placing under a microscope for observation.
The microscopic examination results, 4#, 5#, 6#, 7#, 8#, 9#, 10# and 11# are the same bacterial colony, named as CAMT66351, when observed by an electron microscope, crystal violet is taken as a coloring agent, background color is added for convenient viewing, and the results are observed by an oil lens, and the obtained results are shown in Table 2.
The vegetative hyphae, aerial hyphae and the reproductive hyphae are easy to observe, and the vegetative hyphae grow in the substrate of the culture medium, and the shape of the vegetative hyphae can be observed under a microscope. At the early stage, a plurality of staining agents are adopted as backgrounds, and hyphae observed by taking crystal violet as the staining agent are optimal. Under a microscope, obvious hyphae with a diaphragm can be observed, and the phenomenon that local hyphae are dotted to transparent can be observed. The production process of hyphae is that the spores of filamentous fungi fall on a proper culture medium and germinate and grow. No hypha growing colonies appear in the PDA culture medium, which indicates that the culture medium is not suitable for the growth of the mole cricket cordyceps sinensis endophytic fungi.
TABLE 2 colony morphology
Sixthly, molecular identification of strains
Determining the ITS sequence as shown in SEQ ID NO: 1, and thus, the genus belongs to the phylum Eumycota, subclass Euascomycetes, order Degelatinales, family Clavipitaceae, genus Cordyceps, species Gryllotalpa, anamorphism vismodia himalaica, named Pestalotiopsis vismiae CAMT66351 (abbreviated as Pv-51).
The separated and screened strain CAMT66351 is preserved in Guangdong province microorganism culture Collection (GDMCC), the preservation address is the Guangdong province microorganism research institute, the preservation date is 2016, 12 and 26 days, and the preservation number is GDMCC No: 60135.
EXAMPLE 2 fermentation culture of the Strain
And (3) carrying out liquid fermentation culture on the purified and morphologically stable strain. The potato liquid culture medium and the rice liquid culture medium are adopted for culture. Inoculating pure strains into a test tube liquid culture medium, culturing for 7 days, pouring the culture medium and bacterial colonies in the whole test tube into a liquid triangular flask culture medium, and culturing in a constant-temperature incubator at 20 ℃ to obtain high-yield hyphae, thereby facilitating the later determination of the nutrient content.
The growth effects of the cordyceps endophytic fungi isolates in the three culture media are that: the rice culture medium, the potato culture medium and the PDA culture medium can be designed into a considerable culture medium according to the nutrient content of the formula of the rice culture medium, wherein the growth effect of the rice culture medium is similar to that of the potato culture medium, the colony morphology has no obvious variation, and the culture medium of a natural nitrogen source is good.
Determination of nutrient content by HP L C
The sample preparation comprises grinding mole cricket Cordyceps sinensis endophyte in a mortar, sieving with a 60-mesh sieve, accurately weighing 0.500g sample with an analytical balance, adding water 10m L, performing ultrasonic treatment for 30min, centrifuging, sucking out supernatant, adding 10m L extraction reagent, performing ultrasonic treatment for 30min, centrifuging, mixing supernatants, adding water to constant volume of 25m L, and filtering with 0.45u L microporous membrane for use.
Preparing standard solution by precisely weighing appropriate amounts of inosine, adenosine, uridine and guanosine standard substances, dissolving in a small amount of methanol, diluting with water to constant volume to prepare 0.3mg/m L standard stock solution, and gradually diluting to 0.1, 0.05, 0.02, 0.001 and 0.0005mg/m L standard solution for later use.
The measurement conditions were that the chromatographic column was VP-ODS (5um, phi 4.6 × 250mm), the column temperature was 30 ℃, the injection volume was 10u L, the mobile phase was methanol and water was 75: 25, the flow rate was 1m L/min, and the detection wavelength was 260 nm.
After performing liquid fermentation culture on the CAMT66351, detecting the content of the nucleoside by adopting a high performance liquid chromatography technology. As can be seen from the chromatograms in FIGS. 1-2, under the condition of rice culture medium, the strain CAMT66351 contains a certain amount of four nucleosides, which indicates that the strain has active ingredients of Cordyceps, and may be asexual strain of Cordyceps.
TABLE 3 content determination of four nucleosides by test strain HP L C
SEQUENCE LISTING
<110> Guangdong ocean university
<120> Isaria weberianum CAMT66351 and application thereof
<130>
<160>1
<170>PatentIn version 3.3
<210>1
<211>626
<212>DNA
<213> ITS sequence of CAMT66351
<400>1
tggaaggaaa aaatcgtaac aaggtctccg ttggtgaacc agcggaggga tcattataga 60
gttttctaaa ctcccaaccc atgtgaactt accattgttg cctcggcaga agctgctcgg 120
tgcaccctac cttggaacgg cctaccctgt agcgccttac cctggaacgg cttaccctgt 180
aacggctgcc ggtggactac caaactcttg ttattttatt gtaatctgag cgtcttattt 240
taataagtca aaactttcaa caacggatct cttggttctg gcatcgatga agaacgcagc 300
gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac 360
attgcgccca ttagtattct agtgggcatg cctgttcgag cgtcatttca acccttaagc 420
ctagcttagt gttgggagcc tactgctttt gctagctgta gctcctgaaa tacaacggcg 480
gatctgcgat atcctctgag cgtagtaaat ttttatctcg cttttgactg gagttgcagc 540
gtctttggcc gctaaatccc ccaattttta atggttgacc tcggatcagg taggaatacc 600
cgctgaactt aagcatatca atagtc 626
Claims (8)
1. The pessimios triallatus (Pestalotiopsis vismia) CAMT66351 is preserved in Guangdong province microorganism culture collection center at 2016, 12 and 26 days, with the preservation number being GDMCC No: 60135.
2. the Plasmodium westermanii CAMT66351 according to claim 1, wherein colonies of the fungus grow irregularly, have a dry surface, are milky white, and have spores scattered; with hypha, no septum, and granular spore.
3. The Plasmodium westermanii CAMT66351 according to claim 1, wherein the ITS sequence of the fungus is as shown in SEQ ID NO: 1 is shown.
4. A microbial preparation comprising the wasserpine pestalotiopsis CAMT66351 described in any one of claims 1 to 3.
5. Use of the Desmopsis virescens CAMT66351 of any one of claims 1 to 3 for the production of nucleosides.
6. The use according to claim 5, wherein the nucleoside is adenosine, guanosine, uridine and inosine.
7. A method for producing nucleosides, characterized in that the fungus Plasmodium westermanii CAMT66351 of claim 1 is inoculated into a liquid medium and cultured for a constant period of time at 20 ℃.
8. The method of claim 7, wherein the liquid medium is a rice medium having a composition of: 45g of rice, 20g of glucose, 10g of peptone, 10g of yeast extract powder and KH2PO42g、MgSO42g of water are added until 1000m L is reached, and the pH is adjusted to 5.5.
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| CN101386819A (en) * | 2008-10-28 | 2009-03-18 | 南开大学 | Saprophytic fungus for producing paclitaxel and method for producing paclitaxel using thereof |
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| CN101386819A (en) * | 2008-10-28 | 2009-03-18 | 南开大学 | Saprophytic fungus for producing paclitaxel and method for producing paclitaxel using thereof |
| CN101608165A (en) * | 2009-07-22 | 2009-12-23 | 广西大学 | One strain blazons intends pestalotia bacteria strain and application thereof |
| CN101892165A (en) * | 2010-07-29 | 2010-11-24 | 广西大学 | Widely distributed pestalotia disseminata (thuem.) stey bacterial strain GXSTYJ03 and application thereof |
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