CN109943490B - Preparation and recovery method of Cordyceps guangdongensis protoplast - Google Patents
Preparation and recovery method of Cordyceps guangdongensis protoplast Download PDFInfo
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Abstract
The invention discloses a preparation and recovery method of Cordyceps guangdongensis protoplast. The preparation method comprises the steps of mycelium culture, protoplast preparation, protoplast purification and the like, wherein the protoplast of the cordyceps guangdongensis is obtained by carrying out enzymolysis on the mycelium of the cordyceps guangdongensis by adopting a mixed enzyme system containing lyase and collapsing enzyme, and TB is utilized3And (4) recovering the Cordyceps guangdongensis protoplast by using a recovery culture medium. The invention establishes a Cordyceps guangdongensis protoplast preparation and recovery system for the first time, is beneficial to the implementation of the later-stage genetic transformation research or protoplast fusion technology of Cordyceps guangdongensis, accelerates the research speed of the aspects of inheritance, breeding and the like, is beneficial to promoting the research and application progress of the Cordyceps guangdongensis in the fields of health food and medicine, and benefits the society.
Description
Technical Field
The invention belongs to the field of biology of fungi, and particularly relates to a preparation and recovery method of cordyceps guangdongensis protoplasts.
Background
Cordyceps guangdongensis (Cordyceps guangdongensis) is a new species published in 2008, and a mature fruiting body cultivation system has been successfully domesticated and established. The fruit body is safe and nontoxic, and is officially approved as 'new resource food' (now called 'new food raw material') by the national ministry of health in 2013. Experiments show that the cordyceps guangdongensis sporocarp has obvious pharmacological effects of resisting avian influenza virus, resisting fatigue, prolonging life, treating chronic renal failure, treating chronic bronchitis, regulating immunity and the like. Earlier researches also find that the cordyceps guangdongensis contains rich metabolites including polysaccharide, fatty acid, cordycepic acid, amino acid, adenosine, trace elements and the like. At present, 30 gene clusters participating in the synthesis of secondary metabolites are predicted through genome sequencing and analysis, and the research on the related gene functions of active substances of Cordyceps guangdongensis is still carried out. The invention establishes a preparation and recovery system of Cordyceps guangdongensis protoplast, lays a foundation for realizing gene function research and breeding of Cordyceps guangdongensis, is favorable for deeply understanding the synthesis path of unique metabolites and the functions of key genes, and is also favorable for genetic modification of Cordyceps guangdongensis at the later stage, so that the yield of active metabolites is increased, and more drug lead compounds are obtained.
Protoplasts are the cell parts that are only enveloped by the plasma membrane, and because there is no cell wall, they respond more strongly to inducers and are ideal receptors for genetic engineering. In addition, protoplast fusion can overcome the problem of incompatibility between distant cells, and realize hybridization and gene recombination. The key to protoplast preparation lies in the digestion of the hyphal cell wall, and many factors affecting the yield and quality of protoplasts, such as: the study progress of protoplast method mediated fungal genetic transformation [ J ] plant protection, 2017,43(2): 25-28). Different fungi have enzymolysis systems and enzymolysis conditions suitable for the fungi, and the preparation and recovery of protoplasts are researched in cordyceps sinensis, cordyceps militaris, paecilomyces cicadae and cordyceps alpina at present, however, the preparation method of the protoplast of cordyceps guangdongensis is not established, and the method for preparing the protoplast of cordyceps sinensis by using a mixed enzyme system of lyase and breakdown enzyme is not reported.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of Cordyceps guangdongensis protoplast.
The preparation method of the Cordyceps guangdongensis protoplast comprises the following steps:
s1, mycelium culture: inoculating cordyceps guangdongensis mycelia into a YMPT liquid culture medium for culturing to obtain tender cordyceps guangdongensis mycelia;
s2, preparation of protoplasts: sequentially washing the mycelia with sterile water and KC buffer solution, and adding mixed enzyme solution for enzymolysis to obtain mixed solution containing protoplasts;
s3, purifying protoplasts: and (5) purifying the mixed solution containing the protoplast obtained in the step (S2) to obtain the Cordyceps guangdongensis protoplast.
The total concentration of the enzymes in the mixed enzyme solution of the step S2 is 0.015g/mL, and the mixed enzyme is composed of lyase and collapsing enzyme in a mass ratio of 1: 1.
The crashase CAS number is 85186-71-6 and is purchased from Sigma, and the lyase lyophilized powder commodity number is L1412-5G and is purchased from Sigma.
The enzymolysis in the step S2 is carried out at the rotating speed of 100rpm, the enzymolysis time is 5.5h, and the enzymolysis temperature is 28 ℃.
The formula of the YMPT liquid culture medium is as follows: each liter of culture medium contains 6g of malt extract, 10g of tryptone, 20g of glucose, 6g of yeast extract and deionized water as a solvent.
The formula of the KC buffer solution is as follows: each 300mL of the culture medium contains 13.42g of KCl and CaCl21.665g, deionized water.
The mycelium culture of the step S1 is carried out at 120rpm and 24 ℃ for 12 d.
The purification of step S3 specifically includes: and (4) filtering the mixed solution containing the protoplast obtained in the step (S2) by using sterilized double-layer mirror paper, taking the filtrate, centrifuging the filtrate at 4000rpm and 4 ℃ for 15min, slightly pouring the supernatant to retain the protoplast precipitate, washing the protoplast precipitate by using a KC buffer solution, centrifuging the precipitate at 4000rpm and 4 ℃ for 15min, slightly pouring the supernatant to retain the protoplast precipitate, and obtaining the Cordyceps guangdongensis protoplast.
The second purpose of the invention is to provide a recovery method of Cordyceps guangdongensis protoplast.
The recovery method of Cordyceps guangdongensis protoplasts comprises the following steps: adding KC buffer solution into the Cordyceps guangdongensis protoplast prepared by the above method to obtain protoplast suspension, mixing the suspension with TB3And (4) performing recovery culture in a regeneration culture medium to obtain a regeneration colony.
Said TB3The formula of the regeneration medium is as follows: the culture medium contains 3g of yeast extract, 3g of acid hydrolyzed casein, 200g of sucrose and 15g of low-melting-point agarose per liter of the culture medium, and the solvent is distilled water.
The recovery culture is constant temperature culture at 24 ℃ for 10 days.
The invention has the beneficial effects that: the preparation and recovery system of the cordyceps guangdongensis protoplast is established for the first time, which is beneficial to the implementation of the later genetic transformation research or protoplast fusion and other technologies of the cordyceps guangdongensis, accelerates the research speed of the cordyceps guangdongensis in the aspects of inheritance, breeding and the like, is beneficial to promoting the research and application progress of the cordyceps guangdongensis in the fields of health food and medicine, and benefits the society.
Drawings
FIG. 1 shows the mycelium of Cordyceps guangdongensis.
FIG. 2 shows Cordyceps guangdongensis protoplasts.
FIG. 3 shows the protoplast of Cordyceps guangdongensis in TB3Resuscitated condition on plate.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1 preparation of Cordyceps guangdongensis protoplast
(1) Preparation of a culture medium and a KC buffer solution:
YMPT liquid medium: mixing malt extract 6g, tryptone 10g, glucose 20g, and yeast extract 6g, adding deionized water to constant volume of 1L, sterilizing at 121 deg.C for 20 min.
TB3Regeneration culture medium:taking 3g of yeast extract, 3g of acid hydrolyzed casein, 200g of sucrose and 15g of low-melting point agarose, adding distilled water to a constant volume of 1L, and sterilizing at 121 ℃ for 20 min.
KC buffer solution: collecting KCl 13.42g and CaCl21.665g, adding deionized water to make the volume to 300mL, and sterilizing at 121 ℃ for 20 min.
(2) Preparing and recovering Cordyceps guangdongensis protoplasts:
the cordyceps guangdongensis mycelia are inoculated into 200mL of YMPT liquid medium and cultured at 120rpm and 24 ℃ for 12 days to obtain tender mycelia (mycelia) of cordyceps guangdongensis (FIG. 1). Preparing enzyme solution, adding 0.15g of mixed enzyme of lyase and crashed enzyme (the mass ratio of the lyase to the crashed enzyme is 1:1) into 10mL of KC buffer solution, and filtering and sterilizing by using a disposable sterile filter of 0.22 mu m for later use. 0.5g (about 40) of the young cordyceps guangdongensis mycelia pellet is sucked by a pipette, washed once by sterile water and then washed once by KC buffer solution. Then pouring the prepared enzyme solution into the tender bacteria balls, carrying out enzymolysis for 5.5h at the speed of 100rpm and the temperature of 28 ℃ to obtain a mixed solution containing the protoplast. Filtering the mixed solution with sterilized double-layer mirror paper, collecting filtrate, centrifuging the filtrate at 4000rpm and 4 deg.C for 15min, slightly pouring out supernatant, retaining the protoplast precipitate, and washing the protoplast precipitate with KC buffer solution; centrifuging at 4000rpm and 4 deg.C for 15min, pouring out supernatant, retaining protoplast precipitate, diluting with KC buffer solution, and observing Cordyceps guangdongensis protoplast by microscopic examination (FIG. 2).
Diluting Cordyceps guangdongensis protoplast to 10% with KC buffer solution7Mixing in 45mL of low melting point TB3And (3) in the recovery culture medium, reversing the plate, repeating the three steps, and culturing in a constant-temperature incubator at 24 ℃ for 10 days to obtain mycelia grown by recovery of the protoplast (figure 3).
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (2)
1. A preparation method of Cordyceps guangdongensis protoplasts is characterized by comprising the following steps:
s1, mycelium culture: inoculating cordyceps guangdongensis mycelia into a YMPT liquid culture medium, and culturing at the speed of 120rpm and the temperature of 24 ℃ for 12 days, wherein the formula of the YMPT liquid culture medium is as follows: each liter of culture medium contains 6g of malt extract, 10g of tryptone, 20g of glucose, 6g of yeast extract and deionized water as a solvent, so as to obtain the tender mycelium of the cordyceps guangdongensis;
s2, preparation of protoplasts: washing mycelia with sterile water and a KC buffer solution in sequence, adding a mixed enzyme solution to carry out enzymolysis at a rotating speed of 100rpm for 5.5 hours at an enzymolysis temperature of 28 ℃, wherein the mixed enzyme solution takes the KC buffer solution as a solvent, the total concentration of enzymes in the solution is 0.015g/mL, the mixed enzyme consists of lyase and collapsing enzyme in a mass ratio of 1:1, and a mixed solution containing protoplasts is obtained, wherein the formula of the KC buffer solution is as follows: each 300mL of the culture medium contains 13.42g of KCl and CaCl21.665g, deionized water as solvent;
s3, purifying protoplasts: and (4) filtering the mixed solution containing the protoplast obtained in the step (S2) by using sterilized double-layer mirror paper, taking the filtrate, centrifuging the filtrate at 4000rpm and 4 ℃ for 15min, slightly pouring the supernatant to retain the protoplast precipitate, washing the protoplast precipitate by using a KC buffer solution, centrifuging the precipitate at 4000rpm and 4 ℃ for 15min, slightly pouring the supernatant to retain the protoplast precipitate, and obtaining the Cordyceps guangdongensis protoplast.
2. A recovery method of Cordyceps guangdongensis protoplasts is characterized by comprising the following steps: adding KC buffer solution into the Cordyceps guangdongensis protoplast prepared by the method of preparing Cordyceps guangdongensis protoplast according to claim 1 to obtain a suspension of protoplast, mixing the suspension with TB3Resuscitating and culturing in regeneration medium at 24 deg.C for 10 days to obtain TB3The formula of the regeneration medium is as follows: each liter of culture medium contains 3g of yeast extract, 3g of acid hydrolyzed casein, 200g of sucrose, 15g of low-melting-point agarose and distilled water as a solvent, and a regenerated colony is obtained.
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| CN103215250A (en) * | 2013-03-30 | 2013-07-24 | 徐州鸿宇农业科技有限公司 | Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris |
| CN103266101A (en) * | 2013-04-18 | 2013-08-28 | 天津师范大学 | Fusion method for protoplast of Cordyceps sinensis and Cordyceps militaris |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103215250A (en) * | 2013-03-30 | 2013-07-24 | 徐州鸿宇农业科技有限公司 | Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris |
| CN103266101A (en) * | 2013-04-18 | 2013-08-28 | 天津师范大学 | Fusion method for protoplast of Cordyceps sinensis and Cordyceps militaris |
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