CN109825467A - A kind of efficient cordyceps sinensis isolation of ascospores method - Google Patents

Abstract

The invention discloses a kind of cordyceps sinensis isolation of ascospores methods, it is characterized in that, collect the ascospore that fresh cordyceps sinensis is launched, germination medium is coated on after sterile water dilution, ordinary optical microscope observes ascospore sprouting situation, bright hot spot real-time mark, which is formed by, with microscopes optical path under dark situation environment sprouts single ascospore position, make ring sampler alignment facula position punching sampling by oneself, transfer sprouts monospore to isolation medium culture, obtains single Ascospore development and next purebred aweto.The advantages of this method is simple, accurate, high-efficient, at low cost, achievable first separation operation in average every 90 seconds, success rate can be used for high-volume cordyceps sinensis isolation of ascospores 70% or more, to obtain a large amount of purebred awetos (Ophiocordyceps sinensis).

Description

A kind of efficient cordyceps sinensis isolation of ascospores method

Technical field

The present invention relates to aweto pure culture fields, specifically provide a kind of cordyceps sinensis isolation of ascospores side Method, this method is easy, efficient, at low cost, success rate is high, can be applied to the high-volume separation of cordyceps sinensis ascospore, and institute Obtain aweto be single Ascospore development and come it is purebred, without secondarily purified.

Background technique

Cordyceps sinensis (Cordyceps sinensis) be distributed across Qinghai-Tibet Platean and its surrounding area it is rare it is wild in Medicinal material enjoys great prestige the whole world because it is with high medical value.Its essence be aweto (Ophiocordyceps sinensis) infect the bacterium worm complex that the bat moth larvae development in Alpine meadow is formed.Aweto has important Research and application value.It is successfully listed, and is applied to various already using the Bailing capsule that aweto zymophyte powder is developed Disease treatment has shown huge economic benefit and social benefit.

Usually there are two types of methods for the separation of aweto.One is separate to obtain aweto, the party by tissue Method is relatively simple also relatively conventional, as long as working as surface sterilization processing, has very high probability that can obtain aweto, applies It is relatively broad, but this method has one disadvantage in that, it is absolutely purebred for not can guarantee the aweto of acquisition.There are also a kind of acquisitions The method of cordyceps sinensis be obtained by isolation of ascospores, the disadvantages of the method are as follows, ascospore ejection period is exposed to outer In boundary bad border, ascospore is fragile, surface sterilization processing can not be carried out to it, the aweto speed of growth is extremely slow, it is easy to It is contaminated, although carrying out micromanipulation separation by associated higher instrument can succeed, takes time and effort very much, be not suitable for large quantities of The lock out operation of amount.But the advantages of this method is, once the purebred winter worm that successfully can be obtained single Ascospore development and come Summer grass bacterium.

Therefore be simple and efficient practical cordyceps sinensis isolation of ascospores method aweto separation with answer With significant in research.

Summary of the invention

In view of the drawback in current cordyceps sinensis isolation of ascospores, the present invention provides a kind of cordyceps sinensis being simple and efficient Isolation of ascospores method can work applied to the isolation of ascospores of cordyceps sinensis, in large quantity to obtain single ascus Spore development and come purebred aweto.

The ascospore that the present invention is launched using fresh cordyceps sinensis carries out single sprouting ascospore point as experimental material From to obtain purebred aweto, it is characterised in that comprising steps of

1) it collects ascospore: the Callophane Bag that sterilizes being placed in the fructification in ascospore ejection period and collects ascospore.

2) it sprouts culture: after ascospore is diluted with sterile water, being coated on germination medium, carry out sprouting culture.

3) be separately cultured: microscopically observation spore germination situation, the punching transfer of self-made punching ring are sprouted monospore and are extremely divided It is separately cultured from culture medium.

4) screen aimed strain: bacterium colony upgrowth situation on observation isolation medium, screening meet the winter of representative configuration feature Worm summer grass bacterium.

This method is with the ascospore of sprouting for separation object, is not connect directly to spore during whole operation Touching, without reference to micromanipulation, only by means of an ordinary optical microscope and homemade ring sampler.So this method Have the characteristics that easy, efficient, success rate is high, at low cost, practical, can be applied to large batch of isolation of ascospores work Make.

Below for ease of understanding, description is of the invention in conjunction with specific embodiments.

Detailed description of the invention

Fig. 1 is the primary operational process of this method.

Fig. 2 is the resulting single Ascospore development of this method and growth of the next aweto on isolation medium Situation.

Fig. 3 be morphological feature of the purebred aweto TDB5-2-3 obtained by this method on nutrient growth culture medium and The microscopic features of mycelia.

Fig. 4 is the phylogenetic tree constructed according to the ITS sequence of the resulting purebred aweto TDB5-2-3 of this method, It is aweto that obtained strains are confirmed in terms of molecular biology.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples commercially obtains unless otherwise specified.

Culture medium in following embodiments:

Every 1L solid germination medium is made of following substance: 10g agar and water 1000ml.

Every 1L solid separation culture medium is made of following substance: 200g potato, 20g glucose, 10g peptone, 1g yeast Powder, 15g agar and water.Culture medium is wherein settled to 1000ml with water.

Every 1L solid nutrient medium is made of following substance: 200g potato, 200ml raw milk, 20g glucose, 5g water Solve lactoprotein, 1g yeast powder, 1g potassium dihydrogen phosphate, 0.2g bitter salt, 1 tablet of vitamin B compound, agar 15g and water.Its It is middle that culture medium is settled to 1000ml with water.

The PH nature of above-mentioned culture medium

The preparation method of above-mentioned solid germination medium is as follows: 10g agar weighed in triangular flask, and distilled water 1000ml is added, After 121 DEG C of high pressure steam sterilization 20min, pour into spare in 90mm sterile petri dish.

The preparation method of above-mentioned solid separation culture medium is as follows: will be cut into square 1cm peeled potatoes block and boils 30min, uses The dissolution of other solid reagents is added in filtered through gauze one by one in filtrate, and culture medium is settled to 1000ml with distilled water, and 121 DEG C It is spare that 35mm sterile petri dish is poured into after high pressure steam sterilization 20min.

The preparation method of above-mentioned solid nutrient medium is as follows: will be cut into square 1cm peeled potatoes block and boils 30min, uses The dissolution of other solid reagents is added in filtered through gauze one by one in filtrate, and training is added after raw milk is boiled 10min filtered through gauze It supports in base, culture medium is settled to 1000ml with distilled water, pours into 70mm sterile culture after 121 DEG C of high pressure steam sterilization 20min Ware is spare.

Used main pertinent instruments and reagent have in following embodiments: superclean bench, ordinary optical microscope, perseverance Warm biochemical cultivation case, nucleic acid electrophoresis system, blood counting chamber, self-control ring sampler, OMEGA fungal gene group extraction agent box Deng.

The sprouting culture of embodiment 1, cordyceps sinensis ascospore.

Cordyceps sinensis used in the present invention is the fresh cordyceps sinensis that Tianzhu Area, Gansu area ascospore launches period.It is led Want step are as follows: 1, by sterilize Callophane Bag cover in ascospore ejection period cordyceps sporophore on collect ascospore (Fig. 1 a).2, the ascospore being collected into first is diluted with a small amount of sterile water, it, will after blood counting chamber calculates ascospore density Ascospore suspension exact dilution is to 45/ml.3, liquid-transfering gun is quantitatively drawn 1ml ascospore dilution and is cultivated in 90mm sprouting Base plate is transferred in 15 DEG C of constant incubators after middle spreading rod coating uniformly and carries out sprouting culture, during which constantly seen with microscope Examine ascospore sprouting situation.

Embodiment 2, cordyceps sinensis ascospore are separately cultured

After sprouting culture 3-5 days, ascospore just starts to sprout, and can enter the link being separately cultured.It mainly comprises the following steps 1, 90mm germination medium plate is transferred in superclean bench, and observes ascospore sprouting situation with ordinary optical microscope. 2, after finding single ascospore, confirmed repeatedly with high power lens, sprout monospore (figure to ensure the ascospore in the visual field 1d).3, the fluorescent lamp and all ambient enviroment light sources of superclean bench are closed, to build a dark situation.4, with homemade Microscopes optical path is formed by bright circular light spot (figure on germination medium surface under ring sampler (Fig. 1 b) alignment dark situation 1c) punching sampling.4,35mm isolation medium plate will be seeded to containing sampling block one, one ware form for sprouting monospore In, unification is transferred to 15 DEG C of constant incubators after being sealed with sealed membrane and be protected from light being separately cultured, and routine observation is separately cultured shape Condition.

The nutrient growth culture of embodiment 3, aweto

When being separately cultured about 30 days, the aweto that Ascospore development is individually sprouted in culture dish and is come starts to generate naked eyes Visible growth sign (Fig. 2 a), colonial morphology is obvious (Fig. 2 b) at 60 days, and colony diameter is there are about 1cm or so, and bacterium colony is straight at 90 days Diameter further increases, and pigment is obvious (Fig. 2 c), single Ascospore development has just had successfully been obtained at this time and next cordyceps sinensis Bacterium, if you need to further study with identification can advanced this link of row nutrient growth, key step are as follows: 1, will be by being separately cultured Obtained cordyceps sinensis army is punched with 5mm punch and is sampled, and is inoculated on 70mm nutrient medium plate.2, sealed membrane After sealing, uniformly it is transferred to 18 degrees Celsius of constant incubators and carries out being protected from light nutrient growth culture, and routine observation aweto is sought Upgrowth situation is supported, if it is necessary, photographing to record.

The biological characteristics of embodiment 4, gained aweto

Choose the single Ascospore development that one plant of number on nutrient growth culture medium is TDB5-2-3 and next cordyceps sinensis Bacterial strain carries out biological property research, to ensure correct phorozoon of the obtained bacterial strain as cordyceps sinensis.

1, morphological characteristic: Cordyceps strain bacterium colony on nutrient growth culture medium is strong but pliable in texture, the raw bacterium of initial stage gas Silk is undeveloped or without aerial hyphae, and colony diameter can swell simultaneously up to 3cm or so, entire bacterium colony from intermediate position when cultivating 60 days Marginal portion is spread to, many fold gullies are formed, canescence is integrally presented in bump height about 1CM colony colour, has a small amount of brown Color colouring matter secretion into culture medium (Fig. 3 a)；At bacterium colony culture 90 days or more, aerial hyphae starts to increase and grow nonparasitically upon another plant in entire bacterium Fall surface (Fig. 3 b)；Microscopically observation hyphae colorless is transparent, has interval, compared with branch, part mycelia end can become mycelia Narrow to attenuate and grow kidney shape conidium, conidium is without every conidium is integrally less (Fig. 3 c).

2, molecular biological characteristic: with the third method in OMEGA kit to the total of Cordyceps strain TDB5-2-3 DNA is extracted, and is expanded ribosomes the Internal Transcribed Spacer (ITS) sequence with ITS4/ITS5 universal primer and is sequenced, sequencing result It is committed to NCBI and carries out sequence alignment, obtain kind information, sequencing result is committed to GeneBank and obtains accession number are as follows: MK564659 chooses the wherein high sequence of several similarities (> 99%) phylogenetic tree construction analysis as shown in figure 4, ITS is identified As the result is shown single Ascospore development and come Cordyceps strain be aweto (Ophiocordyceps sinensis).

Claims (4)

1. a kind of cordyceps sinensis isolation of ascospores method, which is characterized in that collect the ascus spore that fresh cordyceps sinensis is launched Son is coated on germination medium after sterile water dilution, ordinary optical microscope observes ascospore sprouting situation, with low light environment Lower microscopes optical path is formed by bright hot spot real-time mark and sprouts single ascospore position, and self-made punching tool punches agar Block obtains single sprouting ascospore, is transferred to isolation medium culture, obtains single Ascospore development and the next purebred winter Worm summer grass bacterium.

2. the advantages of this method be it is simple, accurate, high-efficient, at low cost, can be used for high-volume cordyceps sinensis ascospore point From, with obtain a large amount of purebred awetos (Ophiocordyceps sinensis).

3. application of the separation method described in claim 1 in cordyceps sinensis single spore separation.

4. application of the separation method described in claim 1 in other similar fungi single spore separations.