CN109348990A - A method of improving 808 polyoses content of mushroom kind - Google Patents
A method of improving 808 polyoses content of mushroom kind Download PDFInfo
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- CN109348990A CN109348990A CN201811487515.3A CN201811487515A CN109348990A CN 109348990 A CN109348990 A CN 109348990A CN 201811487515 A CN201811487515 A CN 201811487515A CN 109348990 A CN109348990 A CN 109348990A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
Abstract
The invention discloses a kind of methods for improving 808 polyoses content of mushroom kind, specific steps include: raw material preparation, parent species preparation, original seed preparation, cultivating bag preparation, cultivating bag at 25 DEG C, luminous intensity 0Lx is cultivated to mycelia purseful, and cultivating bag is placed under the conditions of blue light and carries out annesl and management of producing mushroom.The method of the present invention can significantly improve lentinan content, optimize mushroom economical character, improve mushroom production, improve the activity of lignin-degrading enzymes laccase and polysaccharide synthesis key enzyme UGPase enzyme and the expression quantity of correlative coding gene, or optimize existing cultivating champignon mode, mushroom planting benefit, the development offer support of boosting mushroom processing industry are provided.
Description
Technical field
The invention belongs to agricultural plantation technology fields, and in particular to a method of improve 808 polyoses content of mushroom kind.
Background technique
Mushroom (Lentinus edodes) belongs to Basidiomycetes, Agaricales, Pleurotaceae, also known as mushroom, winter wild rice, flower mushroom etc..
Its milpa is mainly distributed on Henan, Fujian, Zhejiang, Anhui, Hunan, Hubei, Jiangxi, Sichuan, Guangdong, Guangxi, Hainan, expensive
The ground such as state, Yunnan, Shaanxi, Gansu.Mushroom is the edible mushroom that current depth a kind of in the world is liked by consumer, in world wide
Output and consumption figure be only second to agaricus bisporus, even more in China's agricultural structure with stronger competitiveness important industry
One of.The mushroom kind that mushroom 808 is promoted mainly as current China, cultivation is most wide, and benefit is best, performance optimal.Mushroom contains more
Kind medicinal ingredient, polysaccharide are a kind of important active substances that mushroom generates, and have antitumor, raising immunity, antiviral, antioxygen
Many-sided function such as change, hypoglycemic, is the main material of current mushroom intensive processing.Since lentinan synthetic quantity is few, extract
Rate is low and expensive, limits it and applies on a large scale, therefore constantly change existing cultivation mode, promotes lentinan content
It is one of the important directions for improving mushroom industrial economy benefit.
There are many factor for influencing lentinan output, including bacterial strain, nutritional ingredient, condition of culture etc..Wherein light is to influence
One key factor of mushroom growth, it will affect morphogenesis, individual and the community distribution of mushroom, film potential variation, physiology
Period, gene expression and annesl formation etc..Therefore, carry out light quality to mushroom annesl, fruit body development, polyoses content shadow
The control that cultivating champignon condition is more rationalized is realized in loud optimal mode application study, this will be helpful to improve lentinan
Content and the batch production development for being pushed further into mushroom industry.
Summary of the invention
The purpose of the present invention is intended to provide a kind of method for improving 808 polyoses content of mushroom kind, plants especially by regulation
The light quality condition of environment is trained to improve 808 polyoses content of mushroom kind, to improve lentinan content and polysaccharide synthesis key enzyme
The expression quantity of enzyme activity and enzyme gene, improve mushroom economical character, improve mushroom production, improve lignin-degrading enzymes laccase activity and
The expression quantity of enzyme gene.
In order to reach above-mentioned technical effect, the present invention is realized especially by following technical scheme:
A method of 808 polyoses content of mushroom kind is improved, specifically includes the following steps:
1) mushroom kind 808 is inoculated in PDA slant medium, carries out strain under the conditions of 25 DEG C of temperature, luminous intensity 0Lx
Activation and parent species preparation；
2) after mycelia covers with inclined-plane, pedigree seed culture medium is loaded after high-temperature sterilization is cooling using green original seeds bottle and connects parent species
Kind, it cultivates under the conditions of 25 DEG C, luminous intensity 0Lx to full bottle；
3) culture is obtained into original seed and is seeded to cultivating bag, cultivating bag makes after filling pedigree seed culture medium and high-temperature sterilization are cooling
With；
4) cultivating bag of inoculation is cultivated under the conditions of 25 DEG C of temperature, illumination 0Lx, after mycelia covers with cultivating bag, will be planted
Training bag is moved to go out to carry out annesl and fruiting in mushroom shed in what is built using blue membrane.
Further, the parent species PDA slant medium are as follows: potato 200g, glucose 20g, agar 20g.
Further, the pedigree seed culture medium includes: sawdust 81%, wheat bran 18%, gypsum according to weight percentage
1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, water content 65%.
Further, the sterilising conditions of the original seeds bottle and cultivating bag are as follows: 0.1MPa, 121 DEG C of sterilizing 2.5h.
Further, the blue membrane goes out mushroom shed using what the film was built using the PVC film of 0.2mm
Intensity of illumination 100Lx, quantum flux density (PPFD) 1.93, blue photon hypothesis (PPF-B) 0.71.
In another aspect of this invention, the method for the present invention can be applied to improve lentinan synthesis key enzyme enzyme activity and its volume
In terms of code controlling gene expression quantity；It applies also for improving mushroom lignin-degrading enzymes laccase activity and its encoding regulator gene table
Up to amount aspect.
It is of the present invention improve lentinan synthesis key enzyme enzyme activity and its encoding regulator gene expression amount in terms of
Using, refer to during lentinan synthesis, is influenced by a series of enzymes, but UGPase (uridine 5'-diphosphate grape
Sugared pyrophosphorylase) it is key enzyme in glycosyl transferase family, the cross-point locations in glycometabolism are in polysaccharide synthesis process
Key enzyme it is a discovery of the invention that blue light can promote the activity and gene expression of the enzyme be a kind of efficiently polysaccharide to be promoted to synthesize
Method.
It is of the present invention to improve the application on lignin-degrading enzymes laccase activity and encoding regulator gene expression amount, refer to
Mushroom needs to decompose lignin in growth and development process to obtain energy matter, and laccase is a kind of important drop of its secretion
The ectoenzyme of lignin is solved, present invention discover that the enzyme enzyme activity and gene expression can be improved in blue light, it is a kind of more efficient using wooden
The method of quality.
The invention has the benefit that
In the inventive solutions, because will affect the multiple beneficials biology such as the annesl of mushroom, fruit-body formation
The light quality of function is strictly regulated and controled and is carried out Field information, and lentinan content can be improved and polysaccharide synthesizes key gene
Expression quantity improves mushroom economical character, improves mushroom production, improves lignin-degrading enzymes laccase activity, not only facilitates optimization
The existing cultivation mode of mushroom promotes mushroom plantation economic benefit, also improves mushroom to the utilization rate of lignin, shows good
Ecological benefits, have applications well prospect.
Specific embodiment
Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
The preparation of 1 mushroom of embodiment, 808 cultivating bag
1.1 parent species preparation and authentications
It selects mushroom to promote mainly kind 808, is inoculated in PDA slant medium (potato 200g, glucose 20g, agar 20g),
25 DEG C are put in, actication of culture and parent species preparation are carried out under the conditions of luminous intensity 0Lx.
The preparation of 1.2 original seeds
Based on 1.1 as a result, after 15d mycelia covers with inclined-plane, start to prepare pedigree seed culture medium (sawdust 81%, wheat bran 18%,
Gypsum 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, water content 65%), use capacity for the green original seeds bottle of 500ml
Charging is sterilized per bottled 800g using high-pressure sterilizing pot, 121 DEG C of sterilizing 2h.After the cooling 12h of original seeds bottle, every parent species are inoculated with
In original seeds bottle, 40 days are cultivated under the conditions of 25 DEG C, luminous intensity 0Lx to spare after full bottle.
The preparation of 1.3 cultivating bags
Based on 1.2 as a result, after original seed covers with bottle, prepare cultivating bag using 14 × 28cm polypropylene plastics pocket, formula with it is former
Kind is identical, and sterilization method is identical with original seeds bottle sterilization method, is inoculated with original seed after cultivating bag is cooling, cultivar is put in 25 DEG C, light
It cultivates under the conditions of intensity 0Lx to purseful.
Mushroom annesl speed and color record under 2 different light medium of embodiment
Mushroom shed is built in field using the black sunshade net of 6 needles.It is oriented and is taken according to illumination, wind direction under mushroom shed out
White (CK), red, five kinds of yellow, green and blue huts are built, each color builds three, and random district's groups arrange, Mei Ge little
1 meter of canopy length, 0.8 meter of width, 0.7 meter of height are spaced 0.8 meter, while holding gutter successfully between hut, it is ensured that and draining is unimpeded, and five
Kind color films select the PVC film of 0.2mm.After 1 shiitake cultivation bag purseful of example to be performed, 20 are placed under each color hut
A cultivating bag.Different light medium processing is as shown in table 1 in mushroom veraison economical character.
Comparison of 1 different light medium of the table processing in mushroom veraison economical character
The quality of mushroom annesl directly affects the performance of later period fruiting, and good annesl effect implies that the production height in later period is of fine quality.
As seen from Table 1, mushroom veraison, different light medium processing significantly affect the annesl color of mycelia annesl speed and mycelia.With regard to annesl
For speed, the mushroom annesl under blue light processing is fastest, handles fast 1.41 times of annesl compared with Conventional white, feux rouges handles annesl
Speed is taken second place；And in terms of the color after annesl, it is best that blue light handles annesl effect.
3 fruiting phase mushroom fruiting body characteristics record of embodiment
For experiment condition with embodiment 2, fruiting phase mushroom fruiting body character is as shown in table 2.
The comparison of 2 different light medium of table processing fructification economic characters
As can be seen from Table 2, different light medium processing is affected to the economic characters of mushroom, blue light processing fruiting time is most fast,
White light takes second place.Blue light processing lower mushroom stem length, stem appropriate diameter, holding stem has hard sense, and bacteria cover diameter is maximum, is
1.02 times of normal white light processing, while cap is most thick and solid, meets high-quality mushroom standard.In addition, blue light handles mushroom production highest,
It is 1.49 times of Conventional white processing.
4 mushroom veraison mycoderma of embodiment and fruiting phase fruitbody polysaccharide assay are measured more using phend-sulphuric acid
Sugared content, the specific steps are as follows: with glucose as a standard product make standard curve.Mycoderma and fructification after weighing 0.2g drying
50 times of volume boiling water are added in liquid powder, and flow back 1h, are repeated 1 times；4 DEG C of sample for flowing back twice, 3000r/min, centrifugation
30min obtains supernatant；4 times of 95% ethyl alcohol of volume of supernatant are added, 4 DEG C of overnight precipitations, are centrifuged by 4 DEG C, 3000r/min
Precipitating is placed in 60 DEG C, removes unnecessary alcohol by 10min；Suitable distilled water dissolution polysaccharide precipitation is added, draws suitable polysaccharide
Solution is in the test tube of wash clean, and moisturizing is to 2mL；The phenol solution of 1mL6% is added, oscillation adds the 5mL concentrated sulfuric acid, fast
Fast shaken well is placed in 25 DEG C of water-bath 30min；Its optical density is measured under 490nm, according to standard curve and institute's densitometric meter
Polyoses content is calculated, the results are shown in Table 3.
Table 3 veraison mycoderma and fruiting phase fruitbody polysaccharide content balance
The results show that blue light processing can significantly improve mushroom mycoderma polyoses content when mushroom mycelium annesl is completed, it is
1.51 times of normal white light processing.Meanwhile blue light processing also increases substantially fructification polyoses content, is normal White-Light processing
1.34 times.
5 polysaccharide of embodiment synthesizes the transcriptional expression analysis of key enzyme enzyme activity and enzyme gene
Veraison mycoderma and fruiting phase fructification, which are cleaned, to be drained, and liquid nitrogen grinding to powder is poured into the centrifuge tube of pre-cooling, added
Enter the extraction buffer of 1mL, 4 DEG C, 12000r/min is centrifuged 20min, and supernatant is crude enzyme liquid.Enzyme activity reaction system is 1mL,
Wherein 960 μ L UGPase enzyme reaction buffer solution, 30 μ L of crude enzyme liquid are eventually adding 10 μ L of 100mmol/L pyrophosphoric acid sylvite, rapidly
Start to react after mixing, the light absorption value under 30 DEG C of reactions 7min, every 30s record 340nm.Form 1 μm of ol's at 30 DEG C per minute
Glucose -1-P is an enzyme activity unit (U), and it is one than living that UGPase Rate activity, which is defined as enzyme activity contained in every mg,
Unit of force.
Extraction RNA reverse transcription is cDNA, and the mushroom UGPase enzyme gene primers announced according to NCBI pass through
RT-qPCR (real-time fluorescence quantitative PCR) analyzes the transcriptional expression difference of key enzyme UGPase gene in polysaccharide synthesis process, uses
2^- △ △ Ct method calculates the relative expression quantity of gene, and the results are shown in Table 4.
4 mycoderma of table and fruitbody polysaccharide key enzyme enzyme activity and gene expression amount comparison
The results show that the UGPase enzyme activity in blue light processing mycoderma reaches 3763 (U/ of highest in mushroom mycelium veraison
Mg), UGPase gene expression amount also improves 1.47 times compared with Conventional white processing.Feux rouges, yellow light and green light all inhibit UGPase
Enzyme enzymatic activity and gene expression amount.In mushroom fruiting phase, blue light handle UGPase enzyme activity in fructification and gene expression amount compared with
Conventional treatment also increases, and enzyme activity reaches 4108 (U/mg), but the not up to level of signifiance.
Embodiment 6 veraison mycoderma and the measurement of fruiting phase mycoderma laccase activity and gene expression amount
2.5g mycoderma-compost mixture is accurately weighed into 250ml triangular flask, 50ml ultrapure water is added and mixes, 25 DEG C,
100rmp oscillation extraction 4h.4 layers of filtered through gauze removing residue, 10,000rmp, 4 DEG C of centrifugation 10min of filtrate, supernatant are as thick
Enzyme solution.Reaction system is by 2.7mL HAc-NaAc buffer, 0.2mL 0.5mmol/L ABTS solution, 0.1mL crude enzyme liquid composition.
It after 25 DEG C of reaction 3min of laccase and substrate, is transferred to rapidly in ice water and terminates reaction, measured with ultraviolet specrophotometer anti-at 420nm
Answer the variation of light absorption value.To boil inactivation crude enzyme liquid as blank control, 3 repetitions of each sample.It defines 1min and aoxidizes 1 μ
Enzyme amount needed for molABTS is an enzyme activity unit (U/mL).ABTS molar extinction coefficient (ε) is 36000mol at 420nm-1·cm-1, then extracting RNA reverse transcription is cDNA, and the catalyzed by laccase from dried mushrooms gene order design primer announced according to NCBI passes through
RT-qPCR (real-time fluorescence quantitative PCR) analyzes the transcriptional expression difference of laccase gene, calculates using using 2^- △ △ Ct method
The relative expression quantity of gene, the results are shown in Table 5.
Table 5 veraison mycoderma and fruiting phase mycoderma laccase activity and gene expression amount
The results show that the mushroom of blue light processing reaches highest 30.33 (U/mg), gene table in veraison laccase enzyme enzymatic activity
Also increase 1.17 times compared with conventional treatment up to amount.The mushroom of blue light processing laccase enzymatic activity also highest 230.66 on fruiting phase mycoderma
(U/mg), but gene expression amount raising is unobvious.
To sum up, mushroom economical character, economic characters, polyoses content, polysaccharide are closed in the processing of overall merit of the present invention light quality
At the influence of key enzyme enzyme activity and gene expression amount, lignin-degrading enzymes laccase activity and gene expression amount.The present invention is aobvious
Show, relative to current common black shading film, in cultivating champignon using blue membrane (i.e. blue light is handled: light intensity 100Lx,
PPFD1.93, PPF-B0.71) polyoses content that can not only significantly improve shiitake mushroom hypha and fructification, perfume (or spice) can also be improved
Mushroom annesl speed and annesl effect, improve mushroom fruiting body economical character, improve polysaccharide synthesis key enzyme UGPase enzyme activity and
Gene expression amount improves lignin-degrading enzymes laccase activity and gene expression amount.Related invention result can be existing to optimize
Cultivating champignon mode improves mushroom planting benefit, the development offer support of boosting mushroom processing industry.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Claims (8)
1. a kind of method for improving 808 polyoses content of mushroom kind, which comprises the following steps:
1) mushroom kind 808 is inoculated in PDA slant medium, carries out actication of culture under the conditions of 25 DEG C of temperature, luminous intensity 0Lx
It is prepared with parent species；
2) after mycelia covers with inclined-plane, pedigree seed culture medium is loaded after high-temperature sterilization is cooling using green original seeds bottle and is inoculated with parent species,
It cultivates under the conditions of 25 DEG C, luminous intensity 0Lx to full bottle；
3) culture is obtained into original seed and is seeded to cultivating bag, cultivating bag uses after filling pedigree seed culture medium and high-temperature sterilization are cooling；
4) cultivating bag of inoculation is cultivated under the conditions of 25 DEG C of temperature, illumination 0Lx, after mycelia covers with cultivating bag, by cultivating bag
It moves to and goes out to carry out annesl and fruiting in mushroom shed in what is built using blue membrane.
2. a kind of method for improving 808 polyoses content of mushroom kind according to claim 1, which is characterized in that described
PDA slant medium are as follows: potato 200g, glucose 20g, agar 20g.
3. a kind of method for improving 808 polyoses content of mushroom kind according to claim 1, which is characterized in that described
Pedigree seed culture medium includes: sawdust 81%, wheat bran 18%, gypsum 1%, potassium dihydrogen phosphate 0.1%, sulfuric acid according to weight percentage
Magnesium 0.05%, water content 65%.
4. a kind of method for improving 808 polyoses content of mushroom kind according to claim 1, which is characterized in that described
The sterilising conditions of original seeds bottle and cultivating bag are as follows: 121 DEG C of sterilizing 2h.
5. a kind of method for improving 808 polyoses content of mushroom kind according to claim 1, which is characterized in that described
Blue membrane using 0.2mm PVC film.
6. a kind of method for improving 808 polyoses content of mushroom kind according to claim 1, which is characterized in that using should
What film was built goes out mushroom shed intensity of illumination 100Lx, quantum flux density 1.93, blue photon hypothesis 0.71.
7. method described in claim 1 is in improving lentinan synthesis key enzyme enzyme activity and its encoding regulator gene expression amount
Application.
8. method described in claim 1 is improving mushroom lignin-degrading enzymes laccase activity and its encoding regulator gene expression amount
In application.
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Cited By (2)
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|---|---|---|---|---|
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| CN113717863A (en) * | 2021-09-03 | 2021-11-30 | 四川农业大学 | Method for increasing 808 polysaccharide content of shiitake variety |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876333A (en) * | 2020-06-30 | 2020-11-03 | 四川农业大学 | Lentinus edodes strain XG-3 capable of producing polysaccharide at high yield and application thereof |
| CN111876333B (en) * | 2020-06-30 | 2022-03-11 | 四川农业大学 | Lentinus edodes strain XG-3 capable of producing polysaccharide at high yield and application thereof |
| CN113717863A (en) * | 2021-09-03 | 2021-11-30 | 四川农业大学 | Method for increasing 808 polysaccharide content of shiitake variety |
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