CN104480188A - Partition type medium group capable of rapidly detecting fungusheterocaryon and preparation method of partition type medium group - Google Patents
Partition type medium group capable of rapidly detecting fungusheterocaryon and preparation method of partition type medium group Download PDFInfo
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- CN104480188A CN104480188A CN201410823677.5A CN201410823677A CN104480188A CN 104480188 A CN104480188 A CN 104480188A CN 201410823677 A CN201410823677 A CN 201410823677A CN 104480188 A CN104480188 A CN 104480188A
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Abstract
A partition type medium group capable of rapidly detecting fungusheterocaryon comprises an inferior medium and an enriching medium and is mounted in one test tube, wherein the inferior medium is located at the lower part of the test tube, the enriching medium is located at the upper part of the test tube, and the inferior medium and the enriching medium are separated by 3-6 high-pressure polypropylene films in the middle; and the inferior medium is mainly prepared from 2-4 g of NH4CL, 2-4 g of NaNO3, 2-3 g of NH4H2PO4, 2-3 g of multivita-glucose, 2-4 g of KH2PO4, 2-4 g of MGSO4, 2-4 g of NaCL, 2-4 g of MnSO4, 2-4 g of CaCL2, 2-4 g of ZnSO4, 1 g of NaHCO3, 150g-300g of potatoes, 20-30g of agar, 10 mg of vitamin B1 and 10mg of vitamin B2. According to the partition type medium group, dominantheterocaryon can be rapidly detected from thousands of variant heterocaryon, the workload for scientific research is reduced, and the working efficiency is improved.
Description
Technical field
Patent of the present invention relates to a kind of microorganism hybridization technique, particularly a kind of substratum group and preparation method thereof of fungi monospore hybridized inheritance Rapid Detection.
Background technology
At present, as everyone knows, utilizing biotechnology to carry out spore hybridization is the effective ways obtaining good quality and high output fungi particularly edible mushrooms new variety, especially particularly common on mushroom industry.
At present, China's edible mushrooms output has become specialty industries in agriculture production, and some places have become and mainstay industry, and the edible mushrooms output of China is primary in the whole world, has become global Edible Fungi big country.
Although China is Edible Fungi big country in the world, but not Edible Fungi power, why say it is not Edible Fungi power, its major cause is that per unit area yield is lower, and what lean on is extensive cultivation, as white mushroom, the output of Holland is every square metre and produces 30 kilograms, and the highest output of China only has 15 kilograms, so technically, China is badly in need of breeding high-yield variety.
Cultivate that high-yield variety is minimum takes 3-5, although and this wherein the monospore hybridization time short, workload is maximum really, and groundwork amount is all used in heterokaryotic detecting.
After monospore hybridization, form thousands of heterokaryon, and in these heterokaryons, according to microbiological genetic development, what positive should make a variation is only a few, majority is that negative should make a variation, and (also the claiming positive energy) of only having positive to make a variation just has the characteristic of high yield and high-quality and strong resistance, and negative should make a variation does not just have these characteristics.
In practice, the heterokaryon that these positives should make a variation be detected, workload is sizable, as looking for a needle in a haystack.
Summary of the invention
Object of the present invention, is to provide a kind of partition-type fungal heterokaryon Rapid Detection substratum group, and the heterokaryon with advantage in the heterokaryon of ten million variation, can detect rapidly, decrease scientific research workload, improve working efficiency by the present invention.
Another object of the present invention, is to provide a kind of preparation method of partition-type fungal heterokaryon Rapid Detection substratum group.
The technical scheme adopted is:
A kind of partition-type fungal heterokaryon Rapid Detection substratum group, is made up of poor general substratum and the rich substratum of increasing, is installed in a test tube.Poor general substratum is positioned at the bottom of test tube, increases the top that rich substratum is positioned at test tube, and centre 3-6 floor height pressure polypropylene film separates.
Described poor general substratum is mainly by NH
4cL 2-4g, NaNO
32-4g, NH
4h
2pO
42-3g, multivita-glucose 2-3g, KH
2pO
42-4g, MGSO42-4g, NaCL 2-4g, MnSO
42-4g, CaCL
22-4g, ZnSO
42-4g, NaHCO3 1g, potato 150-300 g, agar 20-30 g, vitamins B
110mg and vitamins B
210mg is prepared into.
Increase rich substratum mainly by 5% cotton seed hull leach liquor 1000ml, potato 200 g, multivita-glucose 20g, agar 25g, MGSO43g, KH
2pO
43g, NaCL 3g, MnSO
43g, CaCL
23g, ZnSO
43g, peptone 5g, yeast extract paste 6g, vitamins B
110mg, vitamins B
210mg and NaHCO3 1g is prepared into.
Above-mentioned poor general substratum adopts gradient method to develop;
Above-mentioned increasing rich substratum Latin orthogonal experiment method is developed.
The preparation method of poor general substratum, comprises the steps:
1, potato is cleaned and is removed crust, and be cut into the square of 5mm × 5mm by knife, weigh and get 150-300 g, add water to 1000ml, boil 30 minutes, then use 4 layers of filtered through gauze, moisture content, less than 1000ml, adds water to 1000ml, for subsequent use.
2, get potato filtrate 1000 ml prepared by step 1, add 20-30g agar, heating, is stirred to agar and all dissolves, and filter, constant volume 1000 ml, then adds NH in stirring
4cL 2-4g, NaNO
32-4g, NH
4h
2pO
42-3g; , multivita-glucose 2-3g, MGSO4 2-4g, KH
2pO
42-4g, NaCL 2-4g, MnSO
42-4g, CaCL
22-4g ZnSO
43g and NaHCO3 1-1.5g, to be mixed inorganic salt are fully dissolved after, put into autoclave, 120 DEG C, 0.118mpa sterilizing 40 minutes, to obtain final product.
Increase the preparation method of rich substratum, comprise the steps:
1, peeling cleaned by potato, and be cut into the square of 5 × 5mm, get 200 g, add water to 1000 ml, boil 30 minutes, then use 4 layers of filtered through gauze, filtrate adds water to 1000 ml, for subsequent use;
2, get cotton seed hull 50 g, add water 1000 ml, in 60 DEG C of water-soluble pots 60 minutes, filters, get filtrate constant volume 1000 ml, for subsequent use;
3, combining step 1 and step 2 gained filtrate 2000 ml, add agar 25 g, heating, stirs, agar is all dissolved, and filter, constant volume is 1000 ml, then adds MGSO43g, KH
2pO
43g, NaCL 3g, MnSO
43g, CaCL
23g, ZnSO
43g and NaHCO3 1-g, stirs, inorganic salt is all dissolved, then adds peptone 5g, yeast extract paste 6g, vitamins B
110mg, vitamins B
210mg stirs, and 120 DEG C, sterilizing 40 minutes under 0.118mpa, to obtain final product.
Prepare poor general and increase after rich substratum, get the test tube of 20 × 200mm, the one end by bottom loads poor general substratum, piles inclined-plane aseptically, with inoculation rake in the middle of inclined-plane, is excised by top substratum, need be cut into the plane of structure that pendulum is neat.Get sterile polypropylene film 3-6 sheet to be affixed on poor general substratum end face, then implant the rich substratum of increasing with transverse section, be adjacent to, sturdy, obtain partition-type fungal heterokaryon Rapid Detection and cultivate unit.
Advantage of the present invention:
A kind of partition-type fungal heterokaryon Rapid Detection substratum group of the present invention, in the heterokaryon of ten million variation, the heterokaryon with advantage can be detected rapidly, decreases scientific research workload, improve working efficiency, for edible mushrooms rearing new variety creates Liao Yitiaoxin road.
Embodiment
A kind of partition-type fungal heterokaryon Rapid Detection substratum group of the present invention, by the poor general substratum be installed in test tube and the rich substratum of increasing, middle interval establishes high-pressure polypropylene film to form.
Fungi monospore hybridization in, heterokaryotic detecting is the process that workload is larger, and often detect do not know positive relevant variable or negative dependent variable yet, this wherein bears the character of much blindness again.
Poor general substratum of the present invention, will have the switching of heterokaryotic mycelia on this substratum, only have the heterokaryon of the just covert dependent variable of minority to sprout into primary hypha.
The present invention is conducive to that minority in heterokaryon is had detecting of the primary hypha of strong resistance, have and just become after corresponding heterokaryon sprouts on poor general substratum, if at this moment remove picking with inoculating needle, probably meet unintentionally other heterokaryons, can together come by band, for this situation, this invention investigated in test the practice of a cuvette cartridge two kinds of substratum, its test as a result, just becoming corresponding primary hypha, there is certain resistance, poor general substratum also can sprout.
Because newly form heterokaryon, from two parents, and be provided with growth vigor, cell fission speed is fast, in cell in Emp biochemical route, the clean property of enzyme is high, and assimilation is stronger, so in phenotype, mycelia is more vigorous in front end, and major part can be stretched and closely be crossed 3 layer polypropylene films, grows to and increases on rich substratum, on the rich substratum of increasing, primary hyphae kink, forms macroscopic bacterium colony.And those fail the heterokaryon of formation advantage, although also can primary hyphae be formed, but because in the biochemical route such as Emp in cell, the clean property of enzyme is lower, be difficult to cross 3 layer polypropylene films in the very first time, both just define the time difference like this, catch this time difference, picking front end mycelia, the purpose of such work just enhances, and greatly reduces workload.
The research of poor general substratum
According to microbial physiology principle, fungus spore germination needs N source, C source, inorganic salt, growth factor, applicable pH value, according to the difference of formula, be divided into synthetic medium and perfect medium, this poor general cultivation is a kind of substratum in the middle of synthetic medium and perfect medium transition, and spore can be sprouted, need again certain advantage, i.e. strong resistance, adaptable heterokaryon could be sprouted, and the formula of substratum is groped to adopt gradient method to grope to form.
The formula of poor general substratum
By the N source required for fungus spore germination, C source, inorganic salt are divided into three gradients, point No. 1, another name, No. 2, No. 3.
No. 1 substratum
NH
4cL 2g; NaNO
32g; NH
4h
2pO
42g; Multivita-glucose 2g; MGSO4 2g; KH
2pO
42g; NaCL 2g; MnSO
42g; CaCL
22g; ZnSO
42g; NaHCO3 1g
Potato 150g; Agar 20g
Vitamins B
110mg; Vitamins B
210mg
No. 2 culture medium prescriptions
Potato 250g; Agar 25g
NH
4cL 3g; NaNO
33g; NH
4h
2pO
43g; Multivita-glucose 2.5g; MGSO4 3g; KH
2pO
43g; NaCL 3g; MnSO
43g; CaCL
23g; ZnSO
43g; NaHCO3 1g
Vitamins B
110mg; Vitamins B
210mg
No. 3 substratum
Potato 300g; Agar 30g
NH
4cL 4g; NaNO
34g; NH
4h
2pO
43g; Multivita-glucose 3g; MGSO4 4g; KH
2pO
44g; NaCL 4g; MnSO
44g; CaCL
24g; ZnSO
44g; NaHCO3 1g
Vitamins B
110mg; Vitamins B
210mg.
The preparation method of poor general substratum
Potato is cleaned and is removed crust, and be cut into the square of 5mm × 5mm by knife, weigh respectively by formula, add water to 1000ml, boil 30 minutes, then use 4 layers of filtered through gauze, moisture content, less than 1000ml, adds water to 1000ml.
Get filtrate 1000ml, add in filtrate by the agar that takes of each number formula, heat with little fire and stir with glass stick in good time, refilter after agar all dissolves, constant volume 1000ml, and then add other composition respectively, in adition process, want constantly to stir with glass stick, precipitate when preventing from fully not dissolving without salts substances.
By in the test tube of substratum packing 20mm × 200mm after various inorganic salts fully dissolves, fill in tampon, every 51 bundles, wrap with kraft paper, put into autoclave, carry out autoclaving at 120 DEG C of constant-temperature high-pressure 0.118mpa, sterilization time 40 minutes, sterilizing is complete, is put into inclined-plane for subsequent use with regard to heat.
The culture experiment of poor general substratum
Culture vessel: culture vessel made by the culture dish of cut-off footpath 8cm
Materials and methods: get 1,2, No. 3 substratum respectively and do 3 process, each process repeats for 7 times.
Aseptically, substratum is poured in plate, after solidifying, pour 10 into respectively
-5pityrosporion ovale suspension, 24 DEG C of constant temperature 5 days.
Results and analysis:
Constant temperature culture is after 5 days, through data statistic analysis, and No. 1 average spore germination number of substratum 7 bacterium colonies; No. 2 11 bacterium colonies; No. 3 14 bacterium colonies.No. 1 substratum colony number is minimum, and illustrate that No. 1 substratum plays poor general effect, a lot of spore can not be sprouted on this substratum, only has the bacterium colony of relatively strong resistance to sprout, and No. 1 substratum plays the effect of poor general substratum.Can be used as heterokaryon and detect substratum application.
Increase the research of rich substratum
Increasing the effect of rich substratum, is after allowing the mycelia with advantage strong resistance get over polypropylene film isolation strip, climbs to and increases on rich substratum, ramp, displays, so that the mycelium in vitro formed, can with 60 times of magnifying glasses be visible to the naked eye to, be again independently mycelium kink formed.
Increase rich culture medium prescription: the formula increasing rich substratum is groped out through the orthogonal real method of Latin, significantly combine through biometrics differences and be: the cotton seed hull leach liquor 1000ml of 5% replaces moisture content, potato 200g, multivita-glucose 20g, agar 25g, MGSO4 3g; KH
2pO
43g; NaCL 3g; MnSO
43g; CaCL
23g; ZnSO
43g; Peptone 5g; Yeast extract paste 6g; Vitamins B
110mg; Vitamins B
210mg NaHCO3 1g.
Increase the preparation method of rich substratum, comprise the steps:
1, peeling cleaned by potato, and be cut into the square of 5 × 5mm, get 200 g, add water to 1000 ml, boil 30 minutes, then use 4 layers of filtered through gauze, filtrate adds water to 1000 ml, for subsequent use;
2, get cotton seed hull 50 g, add water 1000 ml, in 60 DEG C of water-soluble pots 60 minutes, filters, get filtrate constant volume 1000 ml, for subsequent use;
3, combining step 1 and step 2 gained filtrate 2000 ml, add agar 25 g, heating, stirs, agar is all dissolved, and filter, constant volume is 1000 ml, then adds MGSO43g, KH
2pO
43g, NaCL 3g, MnSO
43g, CaCL
23g, ZnSO
43g and NaHCO3 1-g, stirs, inorganic salt is all dissolved, then adds peptone 5g, yeast extract paste 6g, vitamins B
110mg, vitamins B
210mg stirs, and 120 DEG C, sterilizing 40 minutes under 0.118mpa, to obtain final product.
Prepare poor general substratum and after increasing rich substratum, get the test tube of 20 × 200mm, the one end by bottom loads poor general substratum, aseptically, with inoculation rake in the middle of inclined-plane, is excised by top substratum, needs and the plane of structure becoming pendulum neat.Get sterile polypropylene film 3-6 sheet to be affixed on poor general substratum end face, then implant the rich substratum of increasing with transverse section, be adjacent to, sturdy, obtain partition-type fungal heterokaryon Rapid Detection and cultivate unit.
A kind of preparation method of partition-type fungal heterokaryon Rapid Detection substratum group
Get the test tube of 20 mm × 200mm, load poor general substratum in the one end by bottom, be put into inclined-plane, after inclined-plane is formed, aseptically, with inoculation rake in the middle of inclined-plane, top substratum is cut out, the plane of structure that pendulum is neat need be cut into.
Get the high-pressure polypropylene plastics film in thickness 6 road, be cut into same substratum transverse section shape of the same size, put by polyacrylic film in plastics bag, autoclaving is for subsequent use.
To learn from else's experience autoclaved polypropylene film 3, aseptically, be affixed on poor general substratum section.And then the rich substratum of increasing implanted above with transverse section, to be adjacent to, sturdy, equally also aseptically to carry out, to obtain final product.
Connect bacterium
Aseptically, from the junction surface that A, B two monospores are sprouted on picking plate, the bacterium colony just formed, picking 3 points, are connected on poor general substratum, in 24 DEG C of constant temperature culture with equidistant respectively.
Picking advantage mycelia
After 24 DEG C of constant temperature culture, under 60 times of magnifying glasses, the picking very first time gets over the mycelia that plastics film cuts off, and carries out the examination of later stage screening and the aspect such as biology, cultivation.
Claims (3)
1. a partition-type fungal heterokaryon Rapid Detection substratum group, comprises poor general substratum, increasing rich substratum, polyethylene film and detects test tube, it is characterized in that:
Detecting in test tube, bottom is poor general substratum, and centre is three-layer polypropylene film insulating course, and top is for increasing rich substratum.
2. a kind of partition-type fungal heterokaryon Rapid Detection substratum group according to claim 1, is characterized in that:
Described poor general substratum is mainly by NH
4cL 2-4g, NaNO
32-4g, NH
4h
2pO
42-3g, multivita-glucose 2-3g, KH
2pO
42-4g, MGSO42-4g, NaCL 2-4g, MnSO
42-4g, CaCL
22-4g, ZnSO
42-4g, NaHCO3 1g, potato 150-300 g, agar 20-30 g, vitamins B
110mg and vitamins B
210mg is prepared into;
Increase rich substratum mainly by 5% cotton seed hull leach liquor 1000ml, potato 200 g, multivita-glucose 20g, agar 25g, MGSO43g, KH
2pO
43g, NaCL 3g, MnSO
43g, CaCL
23g, ZnSO
43g, peptone 5g, yeast extract paste 6g, vitamins B
110mg, vitamins B
210mg and NaHCO3 1g is prepared into.
3. the preparation method of a kind of partition-type fungal heterokaryon Rapid Detection substratum group according to claim 1, is characterized in that:
The preparation method of poor general substratum, comprises the steps:
(1) potato is cleaned and is removed crust, and be cut into the square of 5mm × 5mm by knife, weigh and get 150-300 g, add water to 1000ml, boil 30 minutes, then use 4 layers of filtered through gauze, moisture content, less than 1000ml, adds water to 1000ml, for subsequent use;
(2) get potato filtrate 1000 ml prepared by step 1, add 20-30g agar, heating, is stirred to agar and all dissolves, and filter, constant volume 1000 ml, then adds NH in stirring
4cL 2-4g, NaNO
32-4g, NH
4h
2pO
42-3g; , multivita-glucose 2-3g, MGSO4 2-4g, KH
2pO
42-4g, NaCL 2-4g, MnSO
42-4g, CaCL
22-4g ZnSO
43g and NaHCO3 1-1.5g, to be mixed inorganic salt are fully dissolved after, put into autoclave, 120 DEG C, 0.118mpa sterilizing 40 minutes, to obtain final product;
Increase the preparation method of rich substratum, comprise the steps:
(1) peeling cleaned by potato, and be cut into the square of 5 × 5mm, get 200 g, add water to 1000 ml, boil 30 minutes, then use 4 layers of filtered through gauze, filtrate adds water to 1000 ml, for subsequent use;
(2) get cotton seed hull 50 g, add water 1000 ml, in 60 DEG C of water-soluble pots 60 minutes, filters, get filtrate constant volume 1000 ml, for subsequent use;
(3) combining step 1 and step 2 gained filtrate 2000 ml, add agar 25 g, heating, stirs, agar is all dissolved, and filter, constant volume is 1000 ml, then adds MGSO43g, KH
2pO
43g, NaCL 3g, MnSO
43g, CaCL
23g, ZnSO
43g and NaHCO3 1-g, stirs, inorganic salt is all dissolved, then adds peptone 5g, yeast extract paste 6g, vitamins B
110mg, vitamins B
210mg stirs, and 120 DEG C, sterilizing 40 minutes under 0.118mpa, to obtain final product.
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2014
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Patent Citations (4)
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| CN101139593A (en) * | 2007-07-09 | 2008-03-12 | 广东药学院 | House fly antibiotic peptide gene and sifting clone method |
| CN101289670A (en) * | 2008-06-18 | 2008-10-22 | 云南省农业科学院花卉研究所 | Process for obtaining antimycosis cut flower flameray gerbera by transgene |
| CN102342248A (en) * | 2011-09-28 | 2012-02-08 | 金色种业有限公司 | Potato stem apex detoxification breeding method and double-layer culture medium |
| CN104137736A (en) * | 2014-08-08 | 2014-11-12 | 梁平县顶力羊肚菌种植基地 | Method for cultivating morchella esculenta strain |
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Application publication date: 20150401 |