CN111705103B - Method for screening edible fungus variant strain with true positive and no fruiting - Google Patents

Method for screening edible fungus variant strain with true positive and no fruiting Download PDF

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CN111705103B
CN111705103B CN202010645431.9A CN202010645431A CN111705103B CN 111705103 B CN111705103 B CN 111705103B CN 202010645431 A CN202010645431 A CN 202010645431A CN 111705103 B CN111705103 B CN 111705103B
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常金辉
谢逸纯
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Abstract

The invention discloses a method for screening edible fungus variant strains which are true positive and do not fruiting, which comprises the following steps: carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on mycelium after illumination culture, and bacterial colonies with high mycelium growth speed and positive mycelium glycogen staining are primarily screened out; and (3) carrying out fruiting experiments on the bacterial colonies obtained through primary screening, wherein a culture medium in the fruiting experiments contains a GSK-3 activator, and observing that after more than 2 times of fruiting periods, the bacterial colonies are not fruiting, namely, the bacterial colonies are true positive variant strains which are not fruiting. The method for efficiently screening the edible fungus variant strains without fruiting in true positive, which is provided by the invention, solves the problems of lack of obvious screening marks, large workload and high false positive in the early growth stage of the edible fungus variant strains without fruiting in the prior art, and has the characteristics of high accuracy, rapidness, high efficiency and simplicity and convenience in operation.

Description

Method for screening edible fungus variant strain with true positive and no fruiting
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for screening edible fungus variant strains which are true positive and cannot fruiting.
Background
The breeding of edible fungus strains is emphasized, but the breeding research of fruiting body high-yield strains is focused on the majority. Edible fungus mycelia have various applications, such as mycelium powder and polysaccharide extracts for producing edible and medicinal materials as raw materials, and are used for producing mycelium heat insulation boards, mycelium buffer boards and mycelium self-repairable building materials in the field of new materials. However, when the mycelium is changed in temperature, illumination and nutrient substances during the growth process, the nutrient growth path is changed into a reproductive growth path, so that the fruiting body, sclerotium, sporophore and other structural tissues are developed. The transformation of the growth path not only affects the fermentation conversion rate, so that the proportion of active ingredients such as mushroom polysaccharide in fermentation cannot be predicted, but also affects the structural stability of the mycelium material. Therefore, a high-efficiency method is needed to breed strains with only hyphae but not fruiting bodies so as to improve the efficiency and stability of large-scale industrial production.
The conventional edible fungus strain breeding method comprises natural breeding and artificial breeding, wherein the artificial breeding also comprises genetic engineering breeding, hybridization breeding, mutation breeding, protoplast fusion breeding and the like. Conventional identification methods of variant strains obtained by screening breeding comprise morphological identification, biochemical marking and molecular biological identification, but the conventional identification methods have the disadvantages of large workload, long screening period and unstable screened strains. Particularly, when strains which cannot fruiting and are fast in hypha growth are screened, the proportion of ideal strains in the variant strains is low, a large number of false positive strains which cannot fruiting are also present, and when the external environment is slightly changed, fruiting bodies can be regenerated by the strains, so that the loss in large-scale industrial culture is caused.
Furthermore, no report on a method for screening strains against edible fungi which do not fruiting is currently seen.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for screening edible fungus variant strains which are not fruiting in true positive, which solves the problems of lack of obvious screening marks, large workload and high false positive in the early growth period of the fruiting-free strains in the prior art.
The above object of the present invention is achieved by the following means.
A method for screening edible fungus variant strains which are true positive and do not produce mushrooms, which comprises the following steps: carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on the colonies after illumination culture, and colonies with fast hypha growth speed and positive hypha glycogen staining are primarily screened out; and (3) carrying out fruiting experiments on the bacterial colonies obtained through primary screening, wherein a culture medium in the fruiting experiments contains a GSK-3 activator, and observing that after more than 2 times of fruiting periods, the bacterial colonies are not fruiting, namely, the bacterial colonies are true positive variant strains which are not fruiting.
Further, among the colonies which have a high hypha growth rate and are positive in hypha glycogen staining, the colonies which have a high hypha growth rate can be understood as follows: the colony is used as a screening condition, and the colony with the diameter of 1-20 mm, more preferably 5-15 mm; or, taking hypha as a screening condition, the bacterial colony with the growth speed of the hypha in the rapid growth period being more than 4 mm/day; or, it refers to the colony with larger diameter or the colony with higher growth speed of hypha in the same batch, specifically, but not limited to, 1-1000 colonies, preferably 10-1000 colonies, more preferably 10-100 colonies, which are ranked from large to small in diameter or the colony with the growth speed of hypha being ranked from high to low. Colonies whose hyphae are positively stained may be understood to be colonies that appear red, preferably, red, such as their hyphae appear red, reddish brown, etc., more preferably, such as their hyphae appear red other than pink, it being understood that when the hyphae appear pink, the colonies do not go to the next step.
As an improvement of the method for screening the edible fungus variant strains which are not really positive and fruiting, the illumination culture means that illumination is given for not less than 12 hours per day under constant temperature and humidity, and glycogen staining can be carried out after the maximum colony diameter is more than 5mm or the growth speed of the fastest hypha in the rapid growth period is more than 4 mm/day.
As an improvement of the method for screening the edible fungus variant strains which are true positive and do not produce mushrooms, the constant temperature and humidity refers to 10-50 ℃ and 30-100% RH. In particular, the constant temperature and humidity is not limited to the above, and can be adjusted appropriately according to the culture characteristics of different edible fungi, so as to avoid excessive environmental changes, and if the environmental changes are excessive, the growth rate and the metabolic rate can be neglected and slow, so that uniform observation is difficult.
As an improvement of the method for screening the edible fungus variant strains which are not truly positive and fruiting, the light source of illumination is blue light or red-blue light or white light or natural light, such as an incandescent lamp or an LED white light lamp or natural light, and the like.
As an improvement of the method for screening the edible fungus variant strains which are not truly positive and fruiting, the glycogen staining method is a method for staining by using iodine vapor or a periodate glycogen stain.
As an improvement of the method for screening the edible fungus variant strains which are true positive and do not produce mushrooms, the method for fumigating and dyeing by using iodine vapor comprises the following specific steps: taking a beakers without a mouth, uniformly spreading iodine particles on the bottoms of the beakers, heating the beakers covered with a glass plate in a water bath, opening the glass plate after the iodine vapor in the beakers is stable in color, reversely buckling a culture medium growing colonies on the bottle mouth to enable the iodine vapor to contact the whole culture medium, taking down the cover after fumigating for a period of time, and marking colonies with red color in the period of time after fumigating.
As an improvement of the method for screening the edible fungus variant strains which are true positive and not fruiting, the fruiting experiment comprises the following specific steps: filtering a saturated water solution of a GSK-3 activator by a sterile filter, coating the solution on a flat plate culture medium, cutting out bacterial colony blocks which are subjected to primary screening after the surface water is dried, placing the bacterial colony blocks in the center of the flat plate culture medium, and culturing under the illumination condition, wherein each flat plate culture medium is only connected with one single bacterial colony; inducing mushroom under low temperature stimulation, and examining the variant strain which is still fruiting but still not fruiting in more than 2 times of fruiting period.
As an improvement of the method for screening the edible fungus variant strains which are true positive and not fruiting, the fruiting experiment comprises the following specific steps: the strain screened out is activated in an edible fungus culture medium, then is inoculated into an edible fungus cultivation material containing a saturated aqueous solution of a GSK-3 activator, is cultivated under the illumination condition for more than 12 hours at a certain temperature and humidity, after hypha grows fully, the strain is induced under low-temperature stimulation, and the strain is inspected to be a variant strain which is true positive and not fruiting after fruiting in more than 2 times of fruiting period.
It should be noted that GSK-3 activators used above include, but are not limited to, cisplatin, cisplatin.
As an improvement of the method for screening the edible fungus variant strains which are true positive and not fruiting, the variant strains to be screened are variant strains obtained by natural breeding or artificial breeding.
It is noted that the method of the present invention can screen a large number of variant strains obtained by various conventional breeding methods, including but not limited to natural breeding and artificial breeding, and breeding targets including but not limited to genetic variation of protoplasts, spores, sporozoites, hyphae single cells. Wherein, the artificial breeding includes genetic engineering breeding, crossbreeding, mutation breeding (physical or chemical mutation), protoplast fusion breeding and the like.
Specifically, natural breeding is to use natural variation to select desired beneficial variation. Genetic engineering breeding is to copy individual genes of one organism to a new species, modify and transform the genes, put the genes into cells of another organism, and directionally transform the genetic characters of the organism. Mutation breeding is to apply physical or chemical mutagen to the DNA of the strain to make it generate genetic variation to obtain excellent character strain. Crossbreeding is a breeding method for obtaining new characters through exchange and recombination of suitable parent chromosome fragments of two parents so as to obtain the parents with excellent characters. Protoplast fusion is a newly developed cell engineering technology, is a component of cell fusion, and is characterized in that after cell walls are stripped by an enzymatic method, protoplasts of strains with different genetic properties are fused under certain physical or chemical conditions, and genes of the protoplasts and the protoplasts are partially or completely recombined, so that a fusion seed is obtained.
The method of mutation of the mutant strain before screening is not particularly limited, and conventional methods of mutation are available.
As an improvement of the method for screening the edible fungus variant strains which are not truly positive and fruiting, the edible fungus is any one of oyster mushroom, hericium erinaceus, ganoderma lucidum, edible fungus, tremella, flammulina velutipes, grifola frondosa, agaricus blazei, shiitake, agaricus bisporus, agrocybe cylindracea and coprinus comatus. Among them, oyster mushrooms preferably but not limited to include russula rubra, russula flavescens (golden oyster mushroom), white oyster mushroom and black oyster mushroom.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for efficiently screening edible fungus variant strains without fruiting in true positive, which solves the problems of lack of obvious screening marks, large workload and high false positive in the early growth stage of the edible fungus variant strains without fruiting in the prior art.
The invention adopts a two-step screening method to obtain the edible fungus mutant strain with fast hypha growth and no fruiting, and opens up a new way for efficiently screening the strain suitable for hypha fermentation and hypha materials. The operation procedure is simple and convenient, the used instruments and equipment are few, and the method is an edible fungus breeding and screening method which is easy to popularize.
The screening method has short screening period and remarkable effect. The false positive rate of the obtained strain is 0, and one production period is saved in the identification process. And the hereditary character is stable, the hypha character is excellent, the content of active ingredients in hypha is effectively improved, and the economic benefit is remarkably improved.
According to the characteristic of the variation of the junction protein in the signal transduction pathway, the method can be used for rapidly and directionally screening the strain with the specific genetic variation, and a novel screening and identifying method is established. The method can change the current situation that the existing breeding methods of various new varieties lack obvious screening marks, are time-consuming and have large workload, and greatly promote the application and popularization of the edible fungus hyphae in the fields of foods, medicines and new materials.
In the field of new materials, with the technical development of mycelium materials, there is a great need in industry to select strains which cannot fruiting (have no fruiting body) and whose mycelium grows rapidly. In the aspect of food and medicine, mycelium of edible fungi gradually replaces fruit bodies of the edible fungi to become a main raw material for extracting active ingredients, and the production period can be shortened by breeding strains with fast growth of mycelium, and the strains without fruiting can facilitate prediction of the active ingredients of fermentation.
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FIG. 1 is a theoretical basis illustration of a method of screening a true positive non-fruiting variant strain of an edible fungus in accordance with the present invention;
FIG. 2 is a schematic diagram of the method of the present invention for screening a variant strain of an edible fungus that is truly positive and does not produce mushrooms, wherein the shape of the fruiting is merely a schematic state.
Detailed Description
In order to overcome the technical problems described in the background art, the invention provides a method for screening edible fungus variant strains which are true positive and do not fruiting, which comprises the following steps: carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on mycelium after illumination culture, and bacterial colonies with high mycelium growth speed and positive mycelium glycogen staining are primarily screened out; and (3) carrying out fruiting experiments on the bacterial colonies obtained through primary screening, wherein a culture medium in the fruiting experiments contains a GSK-3 activator, and observing that after more than 2 times of fruiting periods, the bacterial colonies are not fruiting, namely, the bacterial colonies are true positive variant strains which are not fruiting.
As shown in fig. 1, the theoretical basis of the screening method is: in fungi, environmental changes such as illumination, temperature, nutrition and the like are converted into chemical signals through receptor proteins, the signals are transmitted through a series of different signal transmission channel proteins and summarized to kinase GSK-3, and only when all the signals can show that the peripheral environment is suitable for fruiting, the kinase GSK-3 is activated, the reproductive growth path is opened, and fruiting bodies and spores start to develop, namely fruiting.
Because of the numerous signal pathways, the number of related proteins and genes is huge, any one of the genetic variations can lead to no fruiting, but the strain which is caused by the variation is usually false positive, namely the strain which is sensitive to the external environment, and once the external environment is changed, the kinase GSK-3 is activated again to start fruiting. Only when the kinase GSK-3 itself or the downstream protein thereof is mutated is the ideal stable fruiting-free strain.
The inventors found that: the ideal stable fruiting-free strain has the following two characteristics besides the characteristic of being capable of keeping stable fruiting-free for a long time: 1) The optical signal can not inhibit the rapid growth of hyphae through the mutated kinase GSK-3, so that the growth speed is faster under the illumination condition compared with the wild strain; 2) Since glycogen synthase cannot be inhibited by the mutated kinase GSK-3 to continue synthesis of glycogen, a large amount of glycogen accumulates in hyphal cells. Based on the method, the growth speed, the morphology and the glycogen dyeing degree of hyphae under the illumination condition are used as primary screening, then the kinase GSK-3 activator is used for inducing the fruiting of false positive strains, and the re-screening of the stable fruiting-free strains is carried out.
The present invention will be described in further detail with reference to specific examples of the specification, but the examples are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Materials, reagents, and the like used in the examples of the present invention are all commercially available.
The following modification methods are merely illustrative, and are not intended to limit the present invention.
Example 1
As shown in FIG. 2, the present example provides a method for screening a truly positive hericium erinaceus variant strain which does not produce mushrooms, wherein the hericium erinaceus variant strain is derived from ultraviolet mutagenesis hericium erinaceus hypha single cells.
The specific steps of ultraviolet mutagenesis of hericium erinaceus hypha single cells are as follows:
(1) preparing hericium erinaceus hypha single cells: taking a double-nucleus Hericium erinaceus PDA inclined plane to store strains, inoculating a small mycelium block into a PDA flat plate culture medium under aseptic condition for culturing for 7 days, cutting 3X 3 mm mycelium block at the front end of the mycelium, transferring into a 2mL centrifuge tube containing small glass beads and 1mL sterile physiological saline under aseptic condition, and vigorously shaking for 10 minutes until the mycelium block is uniformly suspended in the tube; transferring 0.1mL of the clarified liquid into a new 2mL centrifuge tube containing small glass beads and 0.9mL of sterile physiological saline, and vigorously shaking for 3 minutes; repeating dilution and vibration until 20 fields are randomly observed by a microscope at 40 times, wherein hypha is single in distribution, and the phenomenon of aseptic silk polymerization is avoided;
(2) ultraviolet mutagenesis: transferring the hericium erinaceus mycelium single-cell physiological saline solution obtained in the step (1) onto a flat-plate culture dish under aseptic conditions, and irradiating with an ultraviolet lamp under aseptic conditions. Before the ultraviolet mutagenesis treatment, the power supply of the ultra-clean workbench is turned on for 20min, and the ultraviolet lamp light is irradiated after being stabilized. During mutagenesis, the culture dish is placed at a position about 30cm below a 30W ultraviolet lamp of an ultra-clean workbench, a culture dish cover is opened, and the ultraviolet irradiation time is 2 seconds. The duration is adjusted according to the intensity of the ultraviolet lamp, so that the survival rate is 0.1 percent.
The method for screening the hericium erinaceus variant strains which are not fruiting in a true positive way specifically comprises the following steps:
(1) Carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on the colonies after illumination culture, and colonies with fast hypha growth speed and positive hypha glycogen staining are primarily screened out.
Specifically, the variant single cells obtained by ultraviolet mutagenesis are spread on PDA plate culture medium under aseptic condition, so that each plate has 40-60 colonies. Placing at 25deg.C, illuminating with incandescent lamp or LED white light lamp or natural light for not less than 12 hr every day, and culturing with air humidity of 70%. When the diameter of the largest colony in the flat plate is more than 6mm or obviously easy to distinguish, reversely buckling the flat plate above an iodine vapor bottle mouth, fumigating for 15-60 seconds, and then picking out the colony with the diameter of the colony being more than 5mm and the mycelium color being dark brown to reddish brown.
More specifically, glycogen staining specifically proceeds as follows: taking a high-type mouth-free beaker with the height of about 15cm and the diameter of 9cm, uniformly spreading iodine particles on the bottom of the beaker, heating the beaker covered with a glass plate in a water bath with the temperature of 100 ℃, quickly opening the glass plate after iodine vapor in the beaker is stable in color, reversely buckling a flat culture medium growing hyphae on a bottleneck, uniformly contacting the iodine vapor with the whole flat, fumigating for 5-60 seconds, preferably 15-60 seconds, removing a cover, and marking bacterial colonies with red hyphae color within 10 minutes after fumigating. No colorless, off-white or pink color was entered into the fruiting experiments.
(2) And (3) carrying out fruiting experiments on the bacterial colonies obtained through the primary screening, and examining the bacterial colonies which are not fruiting after more than 2 times of fruiting period, namely, the bacterial colonies which are true positive and not fruiting variant strains.
Specifically, after 500 mu L of Cisplatin saturated aqueous solution is filtered by a sterile filter, the solution is coated on a PDA flat plate culture medium, after the surface water is dried, the colony blocks selected in the step (1) are cut and placed in the center of the flat plate for culturing under the illumination condition, and each flat plate is only connected with one single colony; after the hypha grows fully on the flat plate, the mushroom is induced by low-temperature illumination, and the strain which is true and not fruiting is inspected and still does not fruiting in more than 2 times of the conventional fruiting period.
Wherein, the PDA plate culture medium used in the step (1) comprises the following components in percentage by weight: 200g of potato juice, 20g of glucose and KH 2 PO 4 10g，MgSO 4 5g, 20g of agar, 1000mL of water and autoclaving at 121 ℃ for 20 minutes; sterilizing the plate and the mycelium applicator before use; when the PDA culture medium temperature was reduced to 50 ℃, the PDA culture medium was added to 30mL of a plate with a specification of 90mm by 90mm under aseptic conditions, and cooled and solidified.
The hericium erinaceus non-fruiting strain obtained by the screening method of the embodiment has the following index characteristics:
1. effectively shortens the breeding period by more than 50 percent.
2. The beta glucan content is improved by more than 5 percent.
3. The hypha growth speed is improved by more than 10 percent, and the daily growth speed is more than 0.4cm.
4. No fruiting body is produced.
5. The screening method is simple and efficient, and is suitable for industrial production.
Sensory index: the hyphae are white, dense, thick and neat, and the hyphae are microscopic examination to be binuclear hyphae.
Example 2
The embodiment provides a method for screening oyster mushroom variant strains which are not fruiting in true positivity, wherein the oyster mushroom variant strains are from microwave induced oyster mushroom protoplasts.
The specific steps of the microwave induced mushroom protoplast are as follows:
1 strain activation: inoculating the mature mycelium into PDA culture medium, culturing at 28.0+ -0.5deg.C for 40-50h, transferring into PDB mycelium culture solution, culturing at 28.0+ -0.5deg.C and 220rpm for 35-45h;
(2) protoplast preparation: centrifuging the mycelium at 2800rpm for 10min, and collecting the precipitated mycelium; washing with physiological saline for 2 times, adding muramidase with final concentration of 1-2mg/mL, mixing well, and oscillating at 30deg.C for 120min; filtering the enzymolysis liquid, removing fragments such as cell walls and the like, and collecting filtrate; centrifuging the filtrate at 1000rpm for 5min, and precipitating protoplast; washing the protoplast once with normal saline to wash the enzyme solution; discarding the supernatant, and then re-suspending the protoplast with a proper amount of physiological saline;
(3) protoplast microwave treatment: transferring the protoplast bacterial suspension into a sterilized culture dish, performing microwave treatment for 10s in a microwave oven with power of 700W and pulse frequency of 2450Hz, taking out, placing on ice for 20s, and repeating the steps until the accumulated microwave treatment time is 70s; the protoplast is diluted to a dilution of 10 -4 ～10 -7 The dilution was spread on regeneration medium per mL.
A method for screening oyster mushroom variant strains which are true positive and do not fruiting, comprising the following steps:
(1) Carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on mycelium after illumination culture, and bacterial colonies with high mycelium growth speed and positive mycelium PAS staining are primarily screened out.
Specifically, the regeneration culture medium is placed at a constant temperature of 28.0 ℃, and is subjected to illumination for not less than 12 hours per day by an incandescent lamp or an LED white light lamp or natural light, and is cultured for 5-8 days with air humidity of 60%. When the diameter of the largest colony in the culture medium is more than 5mm or is obviously easy to distinguish, PAS glycogen staining is carried out, and then colonies with the diameter of the colony being more than 5mm and positive hyphae PAS staining are picked out.
More specifically, PAS glycogen staining is specifically performed as follows: 1. selecting colony of growth speed block, picking air mycelium on surface, placing on fresh dry smear, fixing with 5% ethanol for 10min, washing with distilled water, and drying; 2. adding saliva or maltose amylase, standing at room temperature for digestion for 60min, and then washing with distilled water; 3.10g/L of periodic acid is oxidized for 15-20 min, washed by distilled water and dried; 4. placing the mixture into a chiffon dye solution for dyeing for 30-60 min at room temperature; 5. washing 3 times with sulfurous acid solution, then washing 2-3 min with tap water, and drying; counterstaining with 6.20g/L of alpha-green for 10-20 min;7. washing with water, drying, and performing microscopic examination. Samples with red cytoplasm and particles were PAS staining positive.
(2) And (3) carrying out fruiting experiments on the bacterial colonies obtained through the primary screening, and examining the bacterial colonies which are not fruiting after more than 2 times of fruiting period, namely, the bacterial colonies which are true positive and not fruiting variant strains.
Specifically, the strain screened out in the first step is activated in a conventional edible fungus culture medium, and then is inoculated into an edible fungus cultivation material containing 1.5% v/v cis-platinum Cisplatin saturated aqueous solution for inducing fruiting, and the variant strain which is true positive and not fruiting is inspected to be fruiting in more than 2 times of conventional fruiting period.
The oyster mushroom non-fruiting strain obtained by the screening method of the embodiment has the following index characteristics:
1. effectively shortens the breeding period by more than 40 percent.
2. The hypha growth speed is improved by more than 10 percent, and the daily growth speed is more than 0.4cm.
3. No fruiting body is produced.
4. The screening method is simple and efficient, and is suitable for industrial production.
Sensory index: the hyphae are white, dense, thick and neat, and the hyphae are microscopic examination to be binuclear hyphae.
Example 3
The embodiment provides a method for screening a ganoderma lucidum variant strain which is true positive and does not produce mushrooms, wherein the ganoderma lucidum variant strain is from molecular breeding.
The molecular breeding is to edit an RNP system by using CRISPR-Cas9 genes and interrupt GSK-3 genes of ganoderma lucidum. CRISPR-Cas9 RNP (ribonucleoprotein complex) is composed of chemically synthesized sgRNA and Cas9 protein, and the chemically synthesized sgRNA is mixed with the Cas9 protein to transfect cells, so that editing of target genes can be rapidly realized. RNP transfection allows optimal dose control of editing complexes, and non-reproducible Cas9 RNPs can be cleared in a short time by endogenous mechanisms, effectively limiting off-target editing. However, RNP transfection has a major limitation in fungal applications due to the lack of a selectable marker. The invention overcomes the difficulty of screening variant strains.
Specific step references for molecular breeding (Jan von, p., escobar, n.,H.A.B.et al.High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins. Sci Rep 9,7632 (2019): https:// doi. Org/10.1038/s 41598-019-44133-2); the simple generalization is as follows:
(1) strain activation: inoculating the mature mycelium into CYM agar culture medium, culturing at 28.0+ -0.5deg.C for 40-50h, transferring into CYM broth culture solution, culturing at 28.0+ -0.5deg.C at 220rpm for 35-45h;
(2) protoplast preparation and RNP preparation: degrading cell walls of mycelia by using muramidase to obtain protoplasts, wherein sorbitol solution is used for keeping osmotic pressure of the protoplasts stable; two chemically synthesized sgrnas for GSK-3 were mixed 2 μg each, and 20 μg Cas9 in 1-fold Cas9 buffer, and incubated at 37 ℃ for 10min to form Cas9 RNP complexes.
(3) PEG-mediated protoplast transfection: 200 μl 2×10 7 Protoplast and Cas9 RNP complex in the presence of CaCl 2 Is mixed in PEG buffer solution. Then spread on CYM agar medium for regeneration.
A method for screening a true positive fruiting-free ganoderma lucidum variant strain specifically comprises the following steps:
(1) Carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on mycelium after illumination culture, and bacterial colonies with high mycelium growth speed and positive mycelium glycogen staining are primarily screened out.
Specifically, the variant single cells obtained by molecular breeding are coated on a CYM flat culture medium under the aseptic condition, so that 40-60 bacterial colonies are formed on each flat plate. Placing at 25deg.C, illuminating with incandescent lamp or LED white light lamp or natural light for not less than 12 hr every day, and culturing with air humidity of 70%. When the diameter of the largest colony in the flat plate is more than 6mm or obviously easy to distinguish, reversely buckling the flat plate above an iodine vapor bottle mouth, fumigating for 15-60 seconds, and then picking out the colony with the diameter of more than 5mm and the color of dark brown to reddish brown.
More specifically, glycogen staining specifically proceeds as follows: taking a high-type mouth-free beaker with the height of about 15cm and the diameter of 9cm, uniformly spreading iodine particles on the bottom of the beaker, heating the beaker covered with a glass plate in a water bath with the temperature of 100 ℃, quickly opening the glass plate after iodine vapor in the beaker is stable in color, reversely buckling a flat culture medium growing hyphae on a bottleneck, uniformly contacting the iodine vapor with the whole flat, fumigating for 5-60 seconds, preferably 15-60 seconds, removing a cover, and marking bacterial colonies with red hyphae color within 10 minutes after fumigating. No colorless, off-white or pink color was entered into the fruiting experiments.
(2) And (3) carrying out fruiting experiments on the bacterial colonies obtained through the primary screening, and examining the bacterial colonies which are not fruiting after more than 2 times of fruiting period, namely, the bacterial colonies which are true positive and not fruiting variant strains.
Specifically, the strain screened initially is activated in a conventional edible fungus culture medium, then is inoculated into an edible fungus cultivation material containing 1.5% v/v Cisplatin (cispratin) saturated aqueous solution for cultivation, after hypha grows up in a material bag, low temperature and light irradiation are given to induce fruiting, and the mutant strain which is true positive and not fruiting is inspected to be fruiting in more than 2 times of conventional fruiting period.
The oyster mushroom non-fruiting strain obtained by the screening method of the embodiment has the following index characteristics:
1. effectively shortens the breeding period by more than 45 percent.
2. The hypha growth speed is improved by more than 10 percent, and the daily growth speed is more than 0.4cm.
3. No fruiting body is produced.
4. The screening method is simple and efficient, and is suitable for industrial production.
Sensory index: the hyphae are white, dense, thick and neat, and the hyphae are microscopic examination to be binuclear hyphae.
Comparative analysis of examples 1, 2, 3 with existing screening methods is performed as follows:
TABLE 1 comparison of screening cycle
TABLE 2 comparison of screening success Rate
As shown in Table 2, the false positive rate of the conventional method in the selected non-fruiting strains is up to 90%, and several rounds of fruiting experiments under different conditions are required. For example, the temperature, nutrition and illumination are changed in different combinations, and even if the method of orthogonal experiment is adopted, fruiting experiment of 3 combinations of conditions is needed. The fruiting experiment needs to consume a large amount of manpower, energy and cultivation materials, the general fruiting period of the edible fungi is generally 0.5 month-6 months, the time cost for one round of experiment is increased, and the cost is high.
According to the invention, as in examples 1, 2 and 3, the number of fruiting experiments is reduced by using the preliminary screening under 2 conditions, the false positive of the fruiting experiments is reduced to 0 by using the GSK-3 activator, the subsequent verification is not needed, and the whole screening process is shortened to only one fruiting period.
According to the method, only 1 fruiting experiment is carried out, the rate of the characteristic variant strains with high hypha growth speed, fruiting failure and the like reaches 50-90%, the workload of the fruiting experiment is reduced (more than 60% of strains subjected to primary screening are fruiting failure strains), the false positive fruiting failure strains are completely removed, and the repeated fruiting experiment time is shortened by 30-45 days.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A method for screening edible fungus variant strains which are true positive and can not produce mushrooms, which is characterized by comprising the following steps: carrying out illumination culture on the variant strains to be screened for a period of time; glycogen staining is carried out on the colonies after illumination culture, and colonies with fast hypha growth speed and positive hypha glycogen staining are primarily screened out; and (3) carrying out fruiting experiments on the bacterial colonies obtained through primary screening, wherein a culture medium in the fruiting experiments contains a GSK-3 activator, and observing that after more than 2 times of fruiting periods, the bacterial colonies are not fruiting, namely, the bacterial colonies are true positive variant strains which are not fruiting.
2. The method for screening a truly positive edible fungus variant strain without fruiting according to claim 1, wherein the light culture means that light is given for not less than 12 hours per day under constant temperature and humidity, and glycogen staining is performed after the maximum colony diameter is 5mm or more or the growth rate of the fastest hypha in the rapid growth period is 4mm/day or more.
3. The method for selecting a variant strain of edible fungus which is true positive and does not produce mushrooms according to claim 2, wherein the constant temperature and humidity is 10-50 ℃ and 30-100% RH.
4. The method for screening a true positive non-fruiting edible fungus variant strain of claim 3 wherein the light source of the illumination is blue light or red blue light or white light or natural light.
5. The method for screening a truly positive, non-fruiting, variant strain of an edible fungus of claim 1 wherein the glycogen staining method is a method of fumigation staining with iodine vapor or staining with a periodate glycogen stain.
6. The method for screening true positive non-fruiting edible fungus variant strains of claim 5 wherein the method for fumigation staining with iodine vapor comprises the specific steps of: taking a beakers without a mouth, uniformly spreading iodine particles on the bottoms of the beakers, heating the beakers covered with a glass plate in a water bath, opening the glass plate after the iodine vapor in the beakers is stable in color, reversely buckling a culture medium growing colonies on the bottle mouth to enable the iodine vapor to contact the whole culture medium, taking down the cover after fumigating for a period of time, and marking colonies with red color in the period of time after fumigating.
7. The method for screening true positive edible fungus variant strains without fruiting according to claim 1, wherein the fruiting experiment comprises the following specific steps: filtering a saturated water solution of a GSK-3 activator by a sterile filter, coating the solution on a flat plate culture medium, cutting out bacterial colony blocks which are subjected to primary screening after the surface water is dried, placing the bacterial colony blocks in the center of the flat plate culture medium, and culturing under the illumination condition, wherein each flat plate culture medium is only connected with one single bacterial colony; after the hypha grows fully, inducing the mushroom under the low-temperature stimulation, and examining the variant strain which is still fruiting in more than 2 times of fruiting period and is true.
8. The method for screening true positive edible fungus variant strains without fruiting according to claim 1, wherein the fruiting experiment comprises the following specific steps: the strain screened out is activated in an edible fungus culture medium, then is inoculated into an edible fungus cultivation material containing a saturated aqueous solution of a GSK-3 activator, is cultivated under the illumination condition for more than 12 hours at a certain temperature and humidity, after hypha grows fully, the strain is induced under low-temperature stimulation, and the strain is inspected to be a variant strain which is true positive and not fruiting after fruiting in more than 2 times of fruiting period.
9. The method for screening true positive edible fungus variant strains without fruiting according to claim 1, wherein the variant strains to be screened are variant strains obtained by natural breeding or artificial breeding.
10. The method according to claim 1, wherein the edible fungus is any one of oyster mushroom, hericium erinaceus, ganoderma lucidum, agaricus, flammulina velutipes, grifola frondosa, agaricus blazei murill, shiitake mushroom, agaricus bisporus, agrocybe cylindracea, and coprinus comatus.
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