CN105219652A - A kind of separation purification method of wild Bachu mushroom mycelium - Google Patents
A kind of separation purification method of wild Bachu mushroom mycelium Download PDFInfo
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Abstract
The invention belongs to wild mushroom hypha separation technical field of purification, be specifically related to a kind of separation purification method of wild Bachu mushroom mycelium, first wild Bachu mushroom tissue or the spore that launches are cultivated on PDA substratum and obtain heterozygosis mycelia in 6-8 days; Then get heterozygosis mycelia and cultivate 7-10 days on Characteristic education base, obtain single thread mycelia of isozygotying; This mycelia of isozygotying of picking is transferred on PDA substratum cultivates 6-8 days.Adopt the DNA after improved method of CTAB extraction purification, and carry out ITS sequence order-checking, the ITS sequence of the sequence obtained and known Bachu mushroom fruitbody is compared, and sequence similarity reaches more than 99%, for artificial culture Bachu mushroom is laid a good foundation.The separation purification method of wild Bachu mushroom mycelium provided by the invention, has that technique is simple, easy handling, purifying are high, purifying is accurate, be easy to the feature of precise Identification.
Description
Technical field
The invention belongs to wild mushroom hypha separation technical field of purification, be specifically related to a kind of separation purification method of wild Bachu mushroom mycelium.
Background technology
Xinjiang Wild domestic fungus resource is relatively less, and famous edible mushrooms, except morel, asafoetida mushroom, also has " the Bachu mushroom " in Kaxgar Prefecture.It is tender delicious, nutritious and famous with matter.Because Bachu mushroom is not tamed successfully up to now, along with people are to the understanding of characteristic fragrance and delicate flavour Bachu mushroom and nutrition, demand increases day by day, market value increases substantially, wild quantity reduces, the living or death of this situation serious threat Bachu mushroom species year by year due to the extinctive collection of local people.
But find through years of researches, Bachu mushroom mycelium purifying difficulty is very big, most mycelia be separated is mixed with shaft-like and heterozygosis mycelia that is sickle shaped two kinds of miscellaneous bacterias, causes research can not continue deeply thus.Find and a kind ofly the novel method of purifying Bachu mushroom mycelium can become the bottleneck of further investigation Bachu mushroom.
So, introduce in traditional hypha separation more effectively, accurately the techniques and methods of wild Bachu mushroom mycelium abstraction and purification be have very great meaning for the preservation of Bachu mushroom species and consumer demand.
Summary of the invention
The object of this invention is to provide a kind of separation purification method of wild Bachu mushroom mycelium, the method is by steps such as wild Bachu mushroom tissue or spore separation cultivation, purifying cultivations, achieve the abstraction and purification of wild Bachu mushroom mycelium, there is purification degrees high, purifying accurately, be easy to the advantage of precise Identification.
Object of the present invention can be achieved through the following technical solutions:
Compared with prior art, beneficial effect of the present invention is:
A separation purification method for wild Bachu mushroom mycelium, its step comprises:
(1), wild Bachu mushroom tissue or the spore that launches are cultivated on PDA substratum within 6-8 days, obtain heterozygosis mycelia;
(2), the heterozygosis mycelia of getting in step (1) cultivates 7-10 days on Characteristic education base, obtains single thread mycelia of isozygotying; Preferably, the culture temperature of this step is 20-25 DEG C.
(3) mycelia, in picking step (2) is transferred on PDA substratum cultivates 6-8 days.
In described step (1), wild Bachu mushroom tissue is the bacterial context of wild Bachu mushroom fruitbody; By bacterial context or the spore that launches, be positioned on the PDA substratum of sterilizing under open environment, under 25 DEG C of conditions, indoor cultivation 7 days.Preferably, the described bacterial context of wild Bachu mushroom fruitbody and the cultivation weight of PDA substratum are 1:90-100; The inoculum size of the spore sterilized water that described wild Bachu mushroom is launched is 0.1-0.5 ‰.After the cultivation of 7 days, there is mycelia to grow, getting the mycelia grown has on the cover glass of distilled water or 5%KOH solution in prior dripping, covered, examines under a microscope mycelia situation, finds that there is three kinds of hypha form, a kind of thread, a kind of sickle shaped, another kind is shaft-like, so, through the cultivation of step (1), the mycelia turned out is heterozygosis mycelia.
Preferably, the bacterial context of described wild Bachu mushroom fruitbody is stem and cap joint portion, and its tissue growth enlivens, and does not contact with extraneous air.
The formula of PDA substratum is potato (peeling) 200g, glucose 20g, agar 15-20g, distilled water 1000mL.
In described step (2), Characteristic education base is made up of glucose, VitB1, gentamicin sulphate, penicillin, Liu Suanyan NEOMYCIN SULPHATE and distilled water.Preferably, the glucose concn of described Characteristic education base is 0.02g/mL; In described Characteristic education base VitB1, gentamicin sulphate, penicillin, Liu Suanyan NEOMYCIN SULPHATE concentration be 0.001mg/mL.
The collocation method of described Characteristic education base is, pours plate into by after glucose, agar and distilled water sterilizing, is cooled to after below 60 DEG C and adds VitB1, gentamicin sulphate, penicillin and Liu Suanyan NEOMYCIN SULPHATE, mixes that to pour flat board into stand-by to solidifying.
The advantage of this Characteristic education base is to suppress the growth of the miscellaneous bacterias such as sickle shaped and shaft-like and bacterium and does not hinder the growth of thread mycelia, the quick growth being convenient to filamentous fungus mycelia be separated.
In described step (2), the inoculum size of heterozygosis mycelia is 1-2 ‰.Suitable heterozygosis mycelium inoculation amount, is conducive to desired mycelia and grows fast, sharp separation.
In described step (3), the inoculum size of mycelia is 1-2 ‰.Select suitable inoculum size again to isozygoty cultivation, the mycelia being conducive to being separated grows faster, recovers its original vigor.
The thread mycelia of isozygotying obtained in step (3) is carried out by microscope inspection and molecular sequence identification qualification of isozygotying, and namely microscopy conforms to sequence alignment purifying, otherwise then impure.
1, the invention provides wild Bachu mushroom mycelium purification process, adopt the DNA after improved method of CTAB extraction purification, and carry out ITS sequence order-checking, the ITS sequence of the sequence obtained and known Bachu mushroom fruitbody is compared, sequence similarity reaches more than 99%, for artificial culture Bachu mushroom is laid a good foundation.
2, the separation purification method of wild Bachu mushroom mycelium provided by the invention, have that purifying is high, purifying accurately, be easy to the feature of precise Identification.
3, wild Bachu mushroom mycelium purifying process of the present invention is simple, easy handling, suitability are strong.
Accompanying drawing explanation
Fig. 1 is the heterozygosis mycelia figure in embodiment 1.
Fig. 2 is the single thread mycelia figure that isozygotys in embodiment 1.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
The preparation of substratum
PDA substratum: potato (peeling) 200g, glucose 20g, agar 15-20g, distilled water 1000mL.
Characteristic education base: glucose, agar and distilled water are configured to 1000mL solution, plate is poured into after sterilizing, being cooled to after below 60 DEG C and adding VitB1 10mg, gentamicin sulphate 10mg, penicillin 10mg and Liu Suanyan NEOMYCIN SULPHATE 10mg, pouring flat board after mixing into stand-by to solidifying.
Purification step:
Sporophore bacterial context (cap and the stem joint portion) 0.1g getting wild Bachu mushroom (plucking from Kashi Bachu County) is incubated in the PDA substratum of 9g, under 25 DEG C of conditions, and indoor cultivation 7 days.
After the cultivation of 7 days, there is mycelia to grow, getting the mycelia grown has on the cover glass of distilled water or 5%KOH solution in prior dripping, and covered, examines under a microscope mycelia situation, find that there is three kinds of hypha form, a kind of thread, a kind of sickle shaped, another kind is shaft-like, so, through the cultivation of above-mentioned steps, the mycelia turned out is heterozygosis mycelia, specifically as shown in Figure 1.
In the Bechtop of sterilizing, picking contains the mycelia block of heterozygosis mycelia, is transferred on Characteristic education base, and inoculum size is cultivate 7-10 days under 1 ‰, 25 DEG C of temperature condition, obtains single thread mycelia of isozygotying.
The above-mentioned single thread mycelia of isozygotying of picking is transferred on PDA substratum and cultivates 7 days, inoculum size is 1 ‰, dripping in advance has on the cover glass of distilled water or KOH solution, and covered, examines under a microscope mycelia situation, find only there is a kind of thread fungus filament shapes, concrete (this thread fungus filament diameter 4-8 μm, slender type, water white transparency as shown in Figure 2, mycelia has barrier film, and mycelia inside has oily drop).
Molecular sequence identification: the mycelia DNA after adopting improved method of CTAB to extract above-mentioned purifying, and carry out ITS order-checking, the ITS sequence of the sequence obtained and known Bachu mushroom fruitbody is compared, and it is then the mycelia of Bachu mushroom that sequence similarity reaches more than 99%.
Mycelia ITS sequence after the purifying measured is as follows.
TAATCAGTTACAGTTGTCTCAGTCAAGAAGACGGTTCGAAGCGGTCGCAAAAACCTTTCACAGAGTCGCATAGGTATCTCTCCAGAGGAGAGAACACAAAGGGAGCCGTGAGGCAAACCCTTCAAACACCCGAGCTCTGCCAACACTCTGAACCAAGCTAGCTTAGATATTATCACAGCTTAGCGTAGCCCAAGTAATGGTTCGTACTGCTAATGCATTTCAGAGGAGCTGAACCCATTGAAAGGTCCGGCAGGCCTCCACCATCCAACACCATTCGAACTCGAAAAACAAGTAAGAAGGGTTGAGAGTTTACTGACACTCAAACAGGCATGCCCTTCGGAATACCAAAGGGCGCAAGGTGCGTTCAAAGACTCGATGATTCACTGAATTCTGCAATTCACATTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGTTGAAAGTTGTATTAAGTTGTTTTAGAGGACGGTGAAGTCCCATAAACGAAGACATTCTAAACATGCAAGAGATGATATGAAGCATAGACCCGGAGGAAACGAAAGGAGGCGAACCTCACCTTCAACCCCAACCAGATCTACAAAAGGTGCACAGGTGGACAAAGAAGAGGCGAGCGACTTCAAAACGTGCACATACTCAAGAGTCAGCAACAGAAGTGAAGCCACAAGCGT
Compare through ITS sequence, sequence similarity reaches more than 99%, and the mycelia that the present invention cultivates is the mycelia of Bachu mushroom.
Embodiment 2
The preparation of substratum
The preparation of PDA substratum and Characteristic education base is with embodiment 1.
Purification step:
Get spore sterilized water 1ml that wild Bachu mushroom launches in PDA substratum, under 25 DEG C of conditions, indoor cultivation 7 days.
After the cultivation of 7 days, there is mycelia to grow, getting the mycelia grown has on the cover glass of distilled water or 5%KOH solution in prior dripping, covered, examine under a microscope mycelia situation, find that there is three kinds of hypha form, a kind of thread, a kind of sickle shaped, another kind is shaft-like, so through the cultivation of above-mentioned steps, the mycelia turned out is heterozygosis mycelia.
In the Bechtop of sterilizing, picking contains the mycelia block of heterozygosis mycelia, is transferred on Characteristic education base, and inoculum size is cultivate 7-10 days under 2 ‰, 25 DEG C of temperature condition, obtains single thread mycelia of isozygotying.
The above-mentioned single thread mycelia of isozygotying of picking is transferred on PDA substratum and cultivates 7 days, inoculum size is 2 ‰, dripping in advance has on the cover glass of distilled water or KOH solution, and covered, examines under a microscope mycelia situation, find only there is a kind of thread fungus filament shapes, this thread fungus filament diameter 4-8 μm, slender type, water white transparency, mycelia has barrier film, and mycelia inside has oily drop.
Molecular sequence identification: the mycelia DNA after adopting improved method of CTAB to extract above-mentioned purifying, and carry out ITS order-checking, the ITS sequence of the sequence obtained and known Bachu mushroom fruitbody is compared, sequence similarity reaches more than 99%, so the mycelia that this preparation method turns out is the mycelia of Bachu mushroom.
The above is preferred embodiment of the present invention, but the present invention should not be confined to the content disclosed in this embodiment.The equivalence completed under not departing from spirit disclosed in this invention so every or amendment, all fall into the scope of protection of the invention.
Claims (7)
1. a separation purification method for wild Bachu mushroom mycelium, its step comprises:
(1), wild Bachu mushroom tissue or the spore that launches are cultivated on PDA substratum within 6-8 days, obtain heterozygosis mycelia;
(2), the heterozygosis mycelia of getting in step (1) cultivates 7-10 days on Characteristic education base, obtains single thread mycelia of isozygotying;
(3) mycelia, in picking step (2) is transferred on PDA substratum cultivates 6-8 days.
2. the separation purification method of wild Bachu mushroom mycelium according to claim 1, is characterized in that: in described step (1), wild Bachu mushroom tissue is the part bacterial context of wild Bachu mushroom fruitbody; By bacterial context or the spore that launches, be positioned on the PDA substratum of sterilizing under open environment, cultivate 7 days under 25 DEG C of conditions.
3. the separation purification method of wild Bachu mushroom mycelium according to claim 2, is characterized in that: the described bacterial context of wild Bachu mushroom fruitbody and the weight of PDA substratum are 1:90-100; The sterilized water inoculum size of the spore that described wild Bachu mushroom is launched is 0.1-0.5 ‰.
4. the separation purification method of wild Bachu mushroom mycelium according to claim 1, it is characterized in that: in described step (2), Characteristic education base is made up of glucose, VitB1, gentamicin sulphate, penicillin, Liu Suanyan NEOMYCIN SULPHATE, distilled water and agar.
5. the separation purification method of wild Bachu mushroom mycelium according to claim 4, is characterized in that: the glucose concn of described Characteristic education base is 0.02g/mL; In described Characteristic education base VitB1, gentamicin sulphate, penicillin, Liu Suanyan NEOMYCIN SULPHATE concentration be 0.001mg/mL.
6. the separation purification method of wild Bachu mushroom mycelium according to claim 1, is characterized in that: in described step (2), the inoculum size of heterozygosis mycelia is 1-2 ‰.
7. the separation purification method of wild Bachu mushroom mycelium according to claim 1, is characterized in that: in described step (3), the inoculum size of described mycelia is 1-2 ‰.
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| CN109055230A (en) * | 2018-07-05 | 2018-12-21 | 上海市农业科学院 | A kind of preparation method of rare medicinal CNNS's Marasmius wild-type strain |
| CN109355200A (en) * | 2018-11-19 | 2019-02-19 | 浙江海洋大学 | A kind of isolation and purification method of the small bolete mycelia of sky handle |
| CN109355201A (en) * | 2018-11-20 | 2019-02-19 | 浙江海洋大学 | A kind of isolation and purification method of the dark red mushroom silk of purple |
| CN110577900A (en) * | 2019-09-25 | 2019-12-17 | 山西农业大学 | strain separation, purification and identification method based on dried Tai mushroom fruiting body |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110577900A (en) * | 2019-09-25 | 2019-12-17 | 山西农业大学 | strain separation, purification and identification method based on dried Tai mushroom fruiting body |
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Inventor after: Li Chuanhua Inventor after: Feng Aiping Inventor after: Zhang Yong Inventor after: Fu Lianjun Inventor after: Wang Jun Inventor after: Wang Haixiao Inventor after: Song Xiaoxia Inventor after: Chen Mingjie Inventor after: Wang Hong Inventor after: Zhang Jinsong Inventor after: Bao Dapeng Inventor after: Shen Xuexiang Inventor after: Xi Liping Inventor before: Li Chuanhua |
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