CN107988203A - Mushroom and matsutake protoplast fusion method - Google Patents
Mushroom and matsutake protoplast fusion method Download PDFInfo
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- CN107988203A CN107988203A CN201711437918.2A CN201711437918A CN107988203A CN 107988203 A CN107988203 A CN 107988203A CN 201711437918 A CN201711437918 A CN 201711437918A CN 107988203 A CN107988203 A CN 107988203A
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- 241000121220 Tricholoma matsutake Species 0.000 title claims abstract description 89
- 235000001674 Agaricus brunnescens Nutrition 0.000 title claims abstract description 54
- 238000007500 overflow downdraw method Methods 0.000 title claims abstract description 28
- 230000002779 inactivation Effects 0.000 claims abstract description 57
- 239000007788 liquid Substances 0.000 claims abstract description 50
- 240000000599 Lentinula edodes Species 0.000 claims abstract description 46
- 235000001715 Lentinula edodes Nutrition 0.000 claims abstract description 40
- 230000004927 fusion Effects 0.000 claims abstract description 30
- 239000001963 growth medium Substances 0.000 claims abstract description 29
- 239000007787 solid Substances 0.000 claims abstract description 28
- 230000008929 regeneration Effects 0.000 claims abstract description 18
- 238000011069 regeneration method Methods 0.000 claims abstract description 18
- 238000005119 centrifugation Methods 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 12
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- 238000005406 washing Methods 0.000 claims abstract description 7
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 29
- 239000008103 glucose Substances 0.000 claims description 28
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- 241000234295 Musa Species 0.000 claims description 12
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
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- 238000012258 culturing Methods 0.000 claims description 3
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- 229930003231 vitamin Natural products 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
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- VSMOENVRRABVKN-UHFFFAOYSA-N oct-1-en-3-ol Chemical compound CCCCCC(O)C=C VSMOENVRRABVKN-UHFFFAOYSA-N 0.000 description 2
- 239000002304 perfume Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- 241000222485 Agaricales Species 0.000 description 1
- 241000272814 Anser sp. Species 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 229920001491 Lentinan Polymers 0.000 description 1
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- 244000046052 Phaseolus vulgaris Species 0.000 description 1
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- 241000121219 Tricholoma Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 229940115286 lentinan Drugs 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 235000004213 low-fat Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000013606 potato chips Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 208000007442 rickets Diseases 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
- C12N15/04—Fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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Abstract
The invention belongs to edible fungus living being breeding technical field, and in particular to a kind of mushroom and matsutake protoplast fusion method, by the The Protoplasts of Lentinus Edodes solution of the purifying of preparation successively through heat inactivation and ultra violet lamp, obtains ultraviolet inactivation The Protoplasts of Lentinus Edodes solution；The matsutake protoplast solution of the purifying of preparation is inactivated through ultra violet lamp and heat successively, obtains heat inactivation matsutake protoplast solution；By ultraviolet inactivation The Protoplasts of Lentinus Edodes solution and heat inactivation matsutake protoplast solution according to 1:1 volume ratio mixing, adds Macrogol 4000, centrifugation, 25 30 DEG C of 10 20min of water-bath, regenerated liquid washing, redissolves in regenerated liquid, obtain recon mixed liquor, be coated on regenerated solids culture medium flat plate, pick out fusion recon.This method Protoplast calli is higher, and the recovery time is short, and regeneration efficiency is high, overcomes the incompatible obstacle in remote source between mushroom and matsutake protoplast category.
Description
Technical field
The invention belongs to edible fungus living being breeding technical field, and in particular to a kind of mushroom and matsutake protoplast fusion side
Method.
Background technology
Mushroom is subordinate to Tricholomataceae, Lentinus, is second-biggest-in-the-world edible mushroom, is known as the title of mountain delicacy.Its delicious flavour, fragrance
Ooze people.Mushroom is full of nutrition, is high protein.The nutrient and healthcare products of low fat, its Quantitative Determination of Ergosterol is very high, to preventing rickets
Effectively, lentinan can improve cell immunocompetent, so as to suppress the growth of cancer cell.The culture conditions of mushroom at present
Highly developed, mild condition, yield are high, cost is low, have high promotional value.
Matsutake is subordinate to Tricholomataceae, Tricholoma, is generation rich in precious material double-strand blazei polysaccharide, matsutake polypeptide and matsutakealcohol
Rare rare natural medicinal fungus, modern medicine study show in boundary, and matsutake has antitumor, anti-aging effects, can improve machine
Body immunity, can treat diabetes, angiocardiopathy, promote functions of intestines and stomach, protection liver.Since matsutake is to growing environment requirement
It is extremely harsh, it there is no method to realize a large amount of complete artificial cultivations at present, only realize Semi-artificial cultivation, low output is expensive.
Therefore the new edible fungus variety that exploitation mushroom is combined with matsutake, for improving edible fungi nutrition value, medical value,
Reduce production cost, raising yield has important economic value.Selection and breeding at present both at home and abroad for edible mushroom new varieties are main
There are monospore partition method, tissue isolation, monofactor mutagenesis, protoplast fusion etc..But mushroom belongs to different from matsutake
Belong to, affiliation farther out, and growing environment difference it is larger, protoplast fusion difficulty is larger, there is no efficient plasm at present
Body fusion method.Chinese patent CN101985617B discloses a kind of straw mushroom and Pleurotus eryngii protoplast fusion method, overcomes
The incompatible obstacle in remote source between section existing for Pleurotus eryngii and straw mushroom, but since straw mushroom belongs to Agaricales, Guang Bing mushrooms section, with matsutake
Fall within and do not belong to together, so straw mushroom and Pleurotus eryngii protoplast fusion method are not particularly suited for melting for mushroom and matsutake protoplast
Close.
To sum up, problem existing in the prior art is to lack a kind of efficient mushroom and matsutake protoplast fusion method.
The content of the invention
To solve the above-mentioned problems, a kind of mushroom and matsutake protoplast fusion method provided by the invention, improve perfume (or spice)
Mushroom and the fusion efficiencies of matsutake protoplast, overcome the incompatible obstacle in remote source between mushroom and matsutake protoplast category.
It is the object of the present invention is to provide a kind of mushroom and matsutake protoplast fusion method, the mushroom of the purifying of preparation is former
Raw plastid solution obtains ultraviolet inactivation The Protoplasts of Lentinus Edodes solution successively through heat inactivation and ultra violet lamp；By the purifying of preparation
Matsutake protoplast solution successively through ultra violet lamp and heat inactivate, obtain heat inactivation matsutake protoplast solution；Will be ultraviolet
The Protoplasts of Lentinus Edodes solution and heat inactivation matsutake protoplast solution are inactivated according to 1:1 volume ratio mixing, adds poly- second two
Alcohol 4000, centrifugation, 25-30 DEG C of water-bath 10-20min, regenerated liquid washing, redissolves in regenerated liquid, obtains recon mixed liquor, apply
It is distributed on regenerated solids culture medium flat plate, picks out fusion recon；
Wherein, the formula of every liter of regenerated liquid is：Mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol, water are settled to 1L；
The formula of every liter of regenerated solids culture medium is：Mashed potatoes 150g, banana puree 50g, glucose 20g, D- sweet dew
Alcohol 0.6mol, agar 20g, water are settled to 1L.
Preferably, above-mentioned mushroom and matsutake protoplast fusion method, specifically includes following steps：
S1, strain culturing
Prepare fresh shiitake mushroom hypha and tricholoma matsutake mycelium；
S2, protoplast electrofusion
The shiitake mushroom hypha collected in S1 is taken, adds mixed enzyme solution, 25-30 DEG C of water enzyme digestion 3-4h, is filtered, and is centrifuged, and is received
Collect The Protoplasts of Lentinus Edodes precipitation, wash, be resuspended, the The Protoplasts of Lentinus Edodes solution purified；The matsutake protoplast of purifying is molten
The preparation method of liquid is identical with the The Protoplasts of Lentinus Edodes solution manufacturing method purified；
S3, the inactivation of protoplast is with merging
By the The Protoplasts of Lentinus Edodes solution of S2 purifying successively through heat inactivation and ultra violet lamp, ultraviolet inactivation mushroom original is obtained
Raw plastid solution；The matsutake protoplast solution of S2 purifying is inactivated through ultra violet lamp and heat successively, obtains heat inactivation matsutake
Protoplast solution；By ultraviolet inactivation The Protoplasts of Lentinus Edodes solution and heat inactivation matsutake protoplast solution according to 1:1 volume
Ratio mixes, and obtains mixed liquor, then adds Macrogol 4000 solution, centrifugation, and 25-30 DEG C of water-bath 10-20min, regenerated liquid is washed
Wash, be resuspended in regenerated liquid, obtain recon mixed liquor, be coated on regenerated solids culture medium flat plate, pick out fusion restructuring
Son.
Preferably, above-mentioned mushroom and matsutake protoplast fusion method, in S3, the formula of every milliliter of mixed enzyme solution
For：Cellulase 10mg, glusulase 10mg, solvent are the epsom salt solution of 0.6mol/L.
Preferably, above-mentioned mushroom and matsutake protoplast fusion method, in S3, heat-inactivated condition is gone out for 65 DEG C of water-bath heat
15min living；The condition of ultra violet lamp is irradiation 10min at 30cm immediately below 30W ultraviolet lamps.
Preferably, above-mentioned mushroom and matsutake protoplast fusion method, in S3, the concentration of Macrogol 4000 solution is
50mmol/L。
Preferably, above-mentioned mushroom and matsutake protoplast fusion method, in S3, the volume of Macrogol 4000 solution is mixed
Close the half of liquid product.
Preferably, above-mentioned mushroom and matsutake protoplast fusion method, in S3, every liter of regenerated liquid is according to following steps
It is prepared：Unpeeled potato is cleaned, is cooked, mashed into paste, obtains mashed potatoes；By banana pulp, banana skin or two
The mixture chopping of person, then smashs to pieces to obtain banana puree；Weigh mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol；It is first that the mashed potatoes weighed, banana puree is soluble in water, 30min is stirred, is filtered, adds what is weighed in obtained filtrate
Glucose and PEARLITOL 25C, are finally settled to 1L with water, obtain regenerated liquid.
Compared with prior art, mushroom and matsutake protoplast fusion method provided by the invention has below beneficial to effect
Fruit：
1st, regenerated liquid and regenerated solids culture medium of the invention are replaced using banana pulp, banana skin or the mixture of the two
Part potato is changed, vitamin is not added and other trace elements only adds steady penetration enhancer PEARLITOL 25C, banana skin cost is low, belongs to useless
The secondary use of thing.To collected after enzymolysis The Protoplasts of Lentinus Edodes precipitation and matsutake protoplast pellet washed, be resuspended and
Regeneration, Protoplast calli is higher, and the recovery time is short, and regeneration efficiency is high, overcomes remote between mushroom and matsutake protoplast category
The incompatible obstacle in source.Experiment is found by contrast, and potato is not removed the peel, and directly cooks use, the regenerated liquid and regenerated solids of preparation
Culture medium it is better.
2nd, ultraviolet inactivation is different with heat-inactivated mechanism, causes the degree of injury of protoplast different, hair is tested through us
Existing, different inactivation modes are merged again after handling, and fusion rate is also different, the inactivation mode of the embodiment of the present invention 1 and fusion side
Method best results.
Embodiment
With reference to specific embodiment, the present invention is described in detail, but should not be construed as the limitation of the present invention.It is following
The test method of actual conditions is not specified in embodiment, is usually operated according to normal condition, due to not being related to inventive point, thus it is not right
Its step is described in detail.
In following embodiments, matsutake Tricholoma mastutake (S.Ito exImai) Sing Shanghai Normal Universitys
" matsutake research " seminar provides, and mushroom Lentinula edodes Pegler, edible mushroom research institute of Shanghai Academy of Agricultural Sciences carries
For.The research that both bacterial strains are merged referring to high wild goose etc., matsutake with The Protoplasts of Lentinus Edodes,《Edible mushroom》2003(2)：9-10.
A kind of mushroom and matsutake protoplast fusion method provided by the invention, by the The Protoplasts of Lentinus Edodes of the purifying of preparation
Solution through heat inactivation and ultra violet lamp, obtains ultraviolet inactivation The Protoplasts of Lentinus Edodes solution successively；By the matsutake of the purifying of preparation
Protoplast solution is inactivated through ultra violet lamp and heat successively, obtains heat inactivation matsutake protoplast solution；Ultraviolet inactivation is fragrant
Mushroom protoplast solution and heat inactivation matsutake protoplast solution are according to 1:1 volume ratio mixing, adds Macrogol 4000,
Centrifugation, 25-30 DEG C of water-bath 10-20min, regenerated liquid washing, redissolves in regenerated liquid, obtains recon mixed liquor, be coated on again
On raw solid medium tablet, fusion recon is picked out；
Wherein, the formula of every liter of regenerated liquid is：Mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol, water are settled to 1L；
The formula of every liter of regenerated solids culture medium is：Mashed potatoes 150g, banana puree 50g, glucose 20g, D- sweet dew
Alcohol 0.6mol, agar 20g, water are settled to 1L.
Including following embodiments.
Embodiment 1
A kind of mushroom and matsutake protoplast fusion method, specifically includes following steps：
S1, strain culturing
Fungus solids culture medium：Potato 20g, glucose 20g, agar 20g, distilled water are settled to 1L.
Fungi liquid culture medium：Potato 200g, glucose 20g, epsom salt 1.5g, potassium dihydrogen phosphate 3g, distilled water
It is settled to 1L.
Conventionally Xianggu mushroom strain is activated using fungus solids culture medium, using fungi liquid culture medium in
The fresh shiitake mushroom hypha of 26 ± 1 DEG C of cultures of triangular flask；Conventionally using fungus solids culture medium to Xianggu mushroom strain into
Row activation, using fungi liquid culture medium in the fresh tricholoma matsutake mycelium of 26 ± 1 DEG C of cultures of triangular flask.
S2, protoplast electrofusion and regeneration
The shiitake mushroom hypha 1g collected in S1 is taken, adds 4mL mixed enzyme solutions, 30 DEG C of water enzyme digestion 3h, funnel filtering, removes
Mushroom mycelium is remained, 3000r/min centrifugation 10min, collect The Protoplasts of Lentinus Edodes precipitation, then wash mushroom original with regenerated liquid
Raw plastid precipitation and 3000r/min centrifugation 10min, repeated washing 2 times, the The Protoplasts of Lentinus Edodes precipitation that last time is collected are resuspended
In regenerated liquid, the The Protoplasts of Lentinus Edodes solution that is purified, the concentration for making The Protoplasts of Lentinus Edodes is 107A/ml, the present embodiment
The concentration of middle The Protoplasts of Lentinus Edodes is 6.9 × 107A/ml, it is spare.
The tricholoma matsutake mycelium 1g collected in S1 is taken, adds 4mL mixed enzyme solutions, 30 DEG C of water enzyme digestion 4h, funnel filtering, removes
Matsutake mycelia is remained, 3000r/min centrifugation 10min, collect matsutake protoplast pellet, then wash matsutake original with regenerated liquid
Simultaneously 3000r/min centrifugations 10min, repeated washing 2 times, the matsutake protoplast pellet that last time is collected are resuspended raw plastid precipitation
In regenerated liquid, the matsutake protoplast solution that is purified, the concentration for making matsutake protoplast is 107A/ml, in the solution
The concentration of matsutake protoplast is 7.5 × 107A/ml, it is spare.
It should be noted that the The Protoplasts of Lentinus Edodes solution of purifying and the matsutake protoplast solution of purifying are coated with respectively
In on regenerated solids culture medium flat plate, 26 ± 1 DEG C of culture 8d, counting counts regenerated protoplast number, calculates plasm respectively
Body regeneration rate, wherein regeneration frequency=(protoplast concentration in regeneration protoplast number/original solution) × 100%, as a result draw,
The regeneration frequency of The Protoplasts of Lentinus Edodes is 7.4%, and matsutake Protoplast calli is 6.9%.
S3, the inactivation of protoplast is with merging
65 DEG C of water-bath heat inactivation 15min of the The Protoplasts of Lentinus Edodes solution purified respectively, obtain heat inactivation The Protoplasts of Lentinus Edodes
Solution, is irradiating 10min by heat inactivation The Protoplasts of Lentinus Edodes solution, is obtaining ultraviolet inactivation perfume at 30cm immediately below 30W ultraviolet lamps
Mushroom protoplast solution；
Respectively the matsutake protoplast solution of S2 purifying is irradiated into 10min at 30cm immediately below 30W ultraviolet lamps, obtain purple
Outer inactivation matsutake protoplast solution；By 65 DEG C of water-bath heat inactivation 15min of ultraviolet inactivation matsutake protoplast solution, obtain heat and go out
Matsutake protoplast solution living；
The inactivation ratio of two protoplast solutions is 100%；
Ultraviolet inactivation The Protoplasts of Lentinus Edodes solution and heat inactivation matsutake protoplast solution are mixed according to each 1ml, obtained mixed
Liquid is closed, then adds the Macrogol 4000 solution of 1ml 50mmol/L, 3000r/min centrifuges 10min, 25 DEG C of water-bath 20min,
Washed 2 times with regenerated liquid, be finally resuspended in regenerated liquid, obtain recon mixed liquor, be coated on regenerated solids culture medium flat plate,
Fusion recon is picked out, calculates fusion rate, fusion rate=(unit volume fusion two parental plant plasm of recon/unit volume
Body sum) × 100%, it is 2.48% that we, which calculate fusion rate, and the recovery time for merging recon is 6d.
In embodiment 1, the formula of every milliliter of mixed enzyme solution is：Cellulase 10mg, glusulase 10mg, solvent are
The epsom salt solution of 0.6mol/L；
The formula of every liter of regenerated liquid is：Mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C 0.6mol,
Water is settled to 1L；Every liter of regenerated liquid is prepared according to following steps：Unpeeled potato is cleaned, cook, is smashed to pieces into
Mud, obtains mashed potatoes；Banana pulp is shredded, then smashs to pieces to obtain banana puree；Weigh mashed potatoes 150g, banana puree 50g, grape
Sugared 20g, PEARLITOL 25C 0.6mol；It is first that the mashed potatoes weighed, banana puree is soluble in water, 30min is stirred, is filtered, obtained filter
The glucose and PEARLITOL 25C weighed is added in liquid, is finally settled to 1L with water, obtains regenerated liquid, sterilizing is spare.
The regenerated solids culture medium is：Mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C 0.6mol, fine jade
Fat 20g, water are settled to 1L；Every liter of regenerated solids culture medium is prepared according to following steps：Unpeeled potato is washed
Only, cook, mashed into paste, obtain mashed potatoes；Banana pulp is shredded, then smashs to pieces to obtain banana puree；Weigh mashed potatoes 150g,
Banana puree 50g, glucose 20g, PEARLITOL 25C 0.6mol, agar 20g；It is first that the mashed potatoes weighed, banana puree is soluble in water, stir
30min is mixed, is filtered, the glucose weighed, PEARLITOL 25C and agar is added in obtained filtrate, is finally settled to 1L with water, boils
Boiling makes agar dissolve to obtain regenerated solids culture medium, and sterilizing is spare.
Embodiment 2
A kind of mushroom and matsutake protoplast fusion method, operation is same as Example 1, mixed difference lies in S2, adding
Synthase liquid, 25 DEG C of water enzyme digestion 4h；In S3, Macrogol 4000 solution, 3000r/min centrifugation 10min, 30 DEG C of water-baths are added
10min；The banana puree that regenerated liquid and regenerated solids culture medium are prepared when preparing using banana skin.Calculating fusion rate is
2.60%, the recovery time for merging recon is 5.5d.
Embodiment 3
A kind of mushroom and matsutake protoplast fusion method, operation is same as Example 1, mixed difference lies in S2, adding
Synthase liquid, 26 DEG C of water enzyme digestion 3.5h；In S3, Macrogol 4000 solution, 3000r/min centrifugation 10min, 26 DEG C of water-baths are added
15min；Using the mixture of banana pulp and banana skin, (direct use is gone when prepared by regenerated liquid and regenerated solids culture medium
The intact banana of stalk) prepare banana puree.It is 2.55% to calculate fusion rate, and the recovery time for merging recon is 6d.
Relevant comparative's example is set forth below, illustrates the effect of the method for the present invention.
First, regenerated liquid effect assessment
The regenerated liquid and regenerated solids culture medium of the present invention is replaced using banana pulp, banana skin or the mixture of the two
Part potato, does not add vitamin and other trace elements only add steady penetration enhancer PEARLITOL 25C, to the mushroom original collected after enzymolysis
Raw plastid precipitation and matsutake protoplast pellet are washed, are resuspended and regenerated, the regeneration of The Protoplasts of Lentinus Edodes in embodiment 1-3
Frequency is 7.4%, 7.1%, 7.2%, and matsutake Protoplast calli is 6.9%, 7.4%, 7.0%, the regeneration of protoplast
Time is 5.5-6d.
The formula of the regenerated liquid of comparative example 1 is：Mashed potatoes 200g, glucose 20g, PEARLITOL 25C 0.6mol, water are settled to
1L；Every liter of regenerated liquid is prepared according to following steps：Unpeeled potato is cleaned, is cooked, mashed into paste, obtains soil
Beans mud；Weigh mashed potatoes 200g, glucose 20g, PEARLITOL 25C 0.6mol；It is first that the mashed potatoes weighed is soluble in water, stirring
30min, filters, and adds the glucose and PEARLITOL 25C weighed in obtained filtrate, is finally settled to 1L with water, is regenerated
Liquid, sterilizing are spare.
The formula of regenerated solids culture medium is：Mashed potatoes 200g, glucose 20g, PEARLITOL 25C 0.6mol, agar 20g, water
It is settled to 1L；Every liter of regenerated solids culture medium is prepared according to following steps：Unpeeled potato is cleaned, is cooked,
Mashed into paste, obtains mashed potatoes；Weigh mashed potatoes 200g, glucose 20g, PEARLITOL 25C 0.6mol, agar 20g；It will first weigh
Mashed potatoes it is soluble in water, stir 30min, filter, add the glucose weighed, PEARLITOL 25C and agar in obtained filtrate,
1L finally is settled to water, boiling makes agar dissolve to obtain regenerated solids culture medium, and sterilizing is spare.
The The Protoplasts of Lentinus Edodes precipitation and matsutake protoplast pellet collected after enzymolysis are washed, are resuspended and regenerated,
The regeneration frequency of The Protoplasts of Lentinus Edodes is 2.2%, and matsutake Protoplast calli is 2.8%, the recovery time of protoplast
For 7d.
The formula of the regenerated liquid of comparative example 2 is：Peeled potatoes piece 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol, water are settled to 1L；Every liter of regenerated liquid is prepared according to following steps：Potato is cleaned, removes the peel section, is obtained
Peeled potatoes piece；Banana pulp is shredded, then smashs to pieces to obtain banana puree；Weigh peeled potatoes piece 150g, banana puree 50g, Portugal
Grape sugar 20g, PEARLITOL 25C 0.6mol；It is first that the potato chips weighed, banana puree is soluble in water, 30min is boiled, filters, obtains
The glucose and PEARLITOL 25C weighed is added in filtrate, is finally settled to 1L with water, obtains regenerated liquid, sterilizing is spare.
The formula of regenerated solids culture medium is：Peeled potatoes piece 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol, agar 20g, water are settled to 1L；Every liter of regenerated solids culture medium is prepared according to following steps：Potato is washed
Only, peeling section, obtains peeled potatoes piece；Banana pulp is shredded, then smashs to pieces to obtain banana puree；Weigh peeled potatoes piece
150g, banana puree 50g, glucose 20g, PEARLITOL 25C 0.6mol, agar 20g；It is first that the peeled potatoes piece weighed, banana puree is molten
Yu Shuizhong, boils 30min, filters, and adds the glucose/PEARLITOL 25C weighed, agar 20g in obtained filtrate, finally uses water
1L is settled to, boiling makes agar dissolve to obtain regenerated liquid, and sterilizing is spare.
The The Protoplasts of Lentinus Edodes precipitation and matsutake protoplast pellet collected after enzymolysis are washed, are resuspended and regenerated,
The regeneration frequency of The Protoplasts of Lentinus Edodes is 3.5%, and matsutake Protoplast calli is 3.1%, the recovery time of protoplast
For 7d.
The result of comparing embodiment 1-3 is with comparative example 1 as a result, finding addition using banana pulp, banana skin or two
The mixture of person replaces part potato, does not add vitamin and other trace elements only add steady penetration enhancer PEARLITOL 25C, plasm
Body regeneration rate is higher, and regeneration efficiency is high.In comparative example 2 processing mode of potato be the prior art conventional treatment mode, through pair
Find that potato do not remove the peel than experiment, directly cook use, the regenerated liquid of preparation and regenerated solids culture medium it is better.
2nd, the contrast test of different inactivation modes
Comparative example 3
The inactivation condition of the The Protoplasts of Lentinus Edodes solution of purifying and the matsutake protoplast solution of purifying is 65 DEG C of water-baths
Heat inactivation 20min.After method according to embodiment 1 is merged, it is 0.68% to calculate fusion rate, merges the regeneration of recon
Time is 7d.
Comparative example 4
The inactivation condition of the The Protoplasts of Lentinus Edodes solution of purifying and the matsutake protoplast solution of purifying is 30W ultraviolet lamps
20min is irradiated at the 30cm of underface.After method according to embodiment 1 is merged, it is 0.55% to calculate fusion rate, fusion weight
The recovery time of group is 7d.
Comparative example 4
The inactivation condition of the The Protoplasts of Lentinus Edodes solution of purifying and the matsutake protoplast solution of purifying is first 65 DEG C of water
Bath heat inactivation 15min, then irradiates 10min at 30cm immediately below 30W ultraviolet lamps.After method according to embodiment 1 is merged,
It is 1.07% to calculate fusion rate, and the recovery time for merging recon is 7d.
The result shows that a variety of inactivation modes are used in combination, different types of protoplast is caused to damage, increase fusion is general
Rate, improves fusion efficiencies, and regenerated liquid promotes protoplast regeneration, shortens the recovery time.In addition, parents strain protoplast into
Row inactivation treatment, can save the screening of genetic marker bacterial strain, be conducive to exclude the interference of the regeneration strain of both sides' parental plant type,
Easy to directly pick out fusion successfully fusion recon.The embodiment of the present invention is correct in order to improve the screening of fusion recon
Rate, screens fusion recon using fluorescent staining method, mushroom parent is carried the fluorchrome of DAPI in advance, make matsutake parent
The fluorchrome of FTTC is carried, then picks out the fusant simultaneous with parents' protoplast fluorescent marker under the microscope.
Different inactivation modes are merged again after handling, and fusion rate is also different, and mainly ultraviolet inactivation is different with heat-inactivated mechanism, leads
Cause the degree of injury of protoplast different, discovery, the inactivation mode and fusion method best results of embodiment 1 are tested through us.
It should be noted that repeat in order to prevent, description of the invention preferred embodiment and effect, although having described
The preferred embodiment of the present invention, but those skilled in the art once know basic creative concept, then can be to these
Embodiment makes other change and modification.So appended claims are intended to be construed to include preferred embodiment and fall into
All change and modification of the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
God and scope.In this way, if these modifications and changes of the present invention belongs to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these modification and variations.
Claims (7)
1. a kind of mushroom and matsutake protoplast fusion method, it is characterised in that the The Protoplasts of Lentinus Edodes of the purifying of preparation is molten
Liquid through heat inactivation and ultra violet lamp, obtains ultraviolet inactivation The Protoplasts of Lentinus Edodes solution successively；The matsutake of the purifying of preparation is former
Raw plastid solution is inactivated through ultra violet lamp and heat successively, obtains heat inactivation matsutake protoplast solution；By ultraviolet inactivation mushroom
Protoplast solution and heat inactivation matsutake protoplast solution are according to 1:1 volume ratio mixing, adds Macrogol 4000, from
The heart, 25-30 DEG C of water-bath 10-20min, regenerated liquid washing, redissolves in regenerated liquid, obtains recon mixed liquor, be coated on regeneration
On solid medium tablet, fusion recon is picked out；
Wherein, the formula of every liter of regenerated liquid is：Mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol, water are settled to 1L；
The formula of every liter of regenerated solids culture medium is：Mashed potatoes 150g, banana puree 50g, glucose 20g, PEARLITOL 25C
0.6mol, agar 20g, water are settled to 1L.
2. mushroom according to claim 1 and matsutake protoplast fusion method, it is characterised in that specifically include following step
Suddenly：
S1, strain culturing
Prepare fresh shiitake mushroom hypha and tricholoma matsutake mycelium；
S2, protoplast electrofusion
The shiitake mushroom hypha collected in S1 is taken, adds mixed enzyme solution, 25-30 DEG C of water enzyme digestion 3-4h, is filtered, centrifugation, is collected fragrant
Mushroom protoplast pellet, is washed, and is resuspended, the The Protoplasts of Lentinus Edodes solution purified；The matsutake protoplast solution of purifying
Preparation method is identical with the The Protoplasts of Lentinus Edodes solution manufacturing method purified；
S3, the inactivation of protoplast is with merging
By the The Protoplasts of Lentinus Edodes solution of S2 purifying successively through heat inactivation and ultra violet lamp, ultraviolet inactivation mushroom plasm is obtained
Liquid solution；The matsutake protoplast solution of S2 purifying is inactivated through ultra violet lamp and heat successively, it is primary to obtain heat inactivation matsutake
Plastid solution；By ultraviolet inactivation The Protoplasts of Lentinus Edodes solution and heat inactivation matsutake protoplast solution according to 1:1 volume ratio
Mixing, obtains mixed liquor, then adds Macrogol 4000 solution, centrifugation, 25-30 DEG C of water-bath 10-20min, regenerated liquid washing, weight
It is suspended from regenerated liquid, obtains recon mixed liquor, be coated on regenerated solids culture medium flat plate, picks out fusion recon.
3. mushroom according to claim 2 and matsutake protoplast fusion method, it is characterised in that in S3, every milliliter of institute
The formula for stating mixed enzyme solution is：Cellulase 10mg, glusulase 10mg, solvent are the epsom salt solution of 0.6mol/L.
4. mushroom according to claim 2 and matsutake protoplast fusion method, it is characterised in that heat-inactivated in S3
Condition is 65 DEG C of water-bath heat inactivation 15min；The condition of ultra violet lamp is irradiation 10min at 30cm immediately below 30W ultraviolet lamps.
5. mushroom according to claim 2 and matsutake protoplast fusion method, it is characterised in that in S3, polyethylene glycol
The concentration of 4000 solution is 50mmol/L.
6. mushroom according to claim 5 and matsutake protoplast fusion method, it is characterised in that in S3, polyethylene glycol
The volume of 4000 solution is the half of mixeding liquid volume.
7. mushroom according to claim 2 and matsutake protoplast fusion method, it is characterised in that in S3, described in every liter
Regenerated liquid is prepared according to following steps：Unpeeled potato is cleaned, is cooked, mashed into paste, obtains mashed potatoes；By banana
Pulp, banana skin or the mixture chopping of the two, then smash to pieces to obtain banana puree；Weigh mashed potatoes 150g, banana puree 50g,
Glucose 20g, PEARLITOL 25C 0.6mol；It is first that the mashed potatoes weighed, banana puree is soluble in water, 30min is stirred, filtering, obtains
Filtrate in add the glucose and PEARLITOL 25C weighed, be finally settled to 1L with water, obtain regenerated liquid.
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| CN110846234A (en) * | 2019-12-23 | 2020-02-28 | 上海市农业科学院 | Pleurotus cornucopiae culture medium |
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