CN101608213A - A kind of discrimination method of hypsizigus marmoreus mating type gene - Google Patents
A kind of discrimination method of hypsizigus marmoreus mating type gene Download PDFInfo
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- CN101608213A CN101608213A CNA2008100390882A CN200810039088A CN101608213A CN 101608213 A CN101608213 A CN 101608213A CN A2008100390882 A CNA2008100390882 A CN A2008100390882A CN 200810039088 A CN200810039088 A CN 200810039088A CN 101608213 A CN101608213 A CN 101608213A
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Abstract
The present invention will obtain sporidium monokaryon bacterial strain from different Hypsizygus marmoreus strains separation, it is carried out mating type gene differentiate.In discrimination process, particularly smooth reaction, fence reaction and the application of transplanting can more tentatively be differentiated hypsizigus marmoreus mating type gene not by Other Instruments and equipment.This method is applied in the cross-breeding, can identifies the mating type gene of Hypsizygus marmoreus parent and filial generation bacterial strain easily, strengthened the purpose and the validity of cross-breeding process.Adopt present method differentiating that the mating type gene time shortens 5～10 days, can make bacterial strain contrast screening quantity reduce 75% during cross-breeding in theory, but improve fecundity, especially at many bacterial strains during repeatedly cross-breeding is used, more convenient efficient.Select affinely then according to identification result, the sporidium monokaryon bacterial strain that clamp connexion occurs matches hybridization, according to the speed of growth, growth potential and economical character index hybrid strain is screened again, and the high yield and high quality hybrid strain is stablized in acquisition.
Description
Technical field:
The present invention relates to the technical field of biological gene,, can improve initiative and validity in Hypsizygus marmoreus list-single crosses breeding specifically according to the discrimination method of Hypsizygus marmoreus sporidium monocaryon mating type gene.
Background technology:
Hypsizygus marmoreus [Hypsizygus marmoreus (Peck) Bigelow], have another name called crab mushroom, seafood mushroom, letter happiness mushroom, beautiful gill fungus, the beautiful mushroom of glue, be under the jurisdiction of Basidiomycotina, Hymenomycetes, Agaricales, white mushroom section, belong to from gill fungus family, beautiful gill fungus, be that a kind of quality is tender and crisp, delicious flavour, nutritious, as to have various health care functions rare edible mushrooms is subjected to liking of consumers in general deeply.
At present, China and some large edible bacterium enterprises of Japan, Korea S can carry out the factory culture Hypsizygus marmoreus by controlled temperature, humidity, illumination and air composition, and obtained good economic benefit, but the bacterial classification that carries out batch production production usefulness is easily degenerated, and occurred catabiosis, brought labile factor to batch production production.Therefore by cross-breeding, selecting the bacterial strain of not only stablizing high yield and high quality but also suitable factory culture is hypsizigus marmoreus in factory production problem demanding prompt solution.And in ongoing cross-breeding process, efficient is generally lower, and effect is also undesirable.Its major cause is that Hypsizygus marmoreus is basidiomycetes such as the different ancestor of standard quadripolarity bonded height, and its mating system is subjected to the control of A and two pairs of mating type genes of B.The polytypism of mating type gene and be the basis of its genetic research and strain improvement to fertility.Do not appear in the newspapers as yet and the discrimination method of relevant hypsizigus marmoreus mating type gene is domestic.
Summary of the invention:
The object of the present invention is to provide a kind of discrimination method of improved hypsizigus marmoreus mating type gene, this method can be stablized the monocaryon that obtains four kinds of standard mating types quickly and accurately from Hypsizygus marmoreus spore colony, improves initiative and validity in Hypsizygus marmoreus list-single crosses breeding process.
To achieve these goals, technical scheme of the present invention is: a kind of discrimination method of hypsizigus marmoreus mating type gene, it is characterized in that: a, obtain sporidium monokaryon bacterial strain from different Hypsizygus marmoreus strains separation, select a Hypsizygus marmoreus spore monokaryon bacterial strain as setting out test strain, be numbered T1, match with all sporidium monokaryon bacterial strains; B, if affine with T1 pairing, and clamp connexion appears, then this sporidium monokaryon bacterial strain is all different with A, the B factor of T1 strain mating type gene; If not affine with the T1 pairing, and smooth reaction appears, then this sporidium monokaryon bacterial strain is different with the identical B factor of the A factor of T1 strain mating type gene; If not affine with the T1 pairing, and the fence reaction appears, then this sporidium monokaryon bacterial strain is identical with the T1 strain mating type gene A factor different B factor; If with the no above-mentioned three kinds of phenomenons of T1 pairing, then this sporidium monokaryon strain mating type gene and T1 strains A, the B factor are all identical.
The present invention will obtain sporidium monokaryon bacterial strain from different Hypsizygus marmoreus strains separation, it is carried out mating type gene differentiate.In discrimination process, particularly smooth reaction, the application of fence reaction and the application of transplanting, the discriminating time shortens 5～10 days, and can more tentatively differentiate hypsizigus marmoreus mating type gene not by Other Instruments and equipment.Utilized the character of double-core strain growth speed faster than the monokaryon bacterial strain among the present invention, the method that utilization is transplanted is amplified this character, can significantly four kinds of mating situations be distinguished in the short period.This method is applied in the cross-breeding, can identifies the mating type gene of Hypsizygus marmoreus parent and filial generation bacterial strain easily, strengthened the purpose and the validity of cross-breeding process.Adopt present method can make bacterial strain contrast screening quantity reduce 75% in theory, but improve fecundity, especially at many bacterial strains during repeatedly cross-breeding is used, more convenient efficient.Select affinely then according to identification result, the spore monokaryon bacterial strain that clamp connexion occurs matches hybridization, according to the speed of growth, growth potential and economical character index hybrid strain is screened again, and the high yield and high quality hybrid strain is stablized in acquisition.
Description of drawings:
Fig. 1 is the process flow sheet of the invention process process
Fig. 2 is a clamp connexion process synoptic diagram of the present invention
Fig. 3 is a smooth reaction bacterium colony photo of the present invention
Fig. 4 is a fence reaction bacterium colony photo of the present invention
Fig. 5 is the bacterium colony photo of two affine bacterial strains of the present invention
Fig. 6 is two the identical bacterial strain bacterium colony of mating type gene photos of the present invention
Embodiment:
The invention will be further described below in conjunction with drawings and Examples.
A kind of discrimination method of hypsizigus marmoreus mating type gene, its difference with the prior art is: a, obtain sporidium monokaryon bacterial strain from different Hypsizygus marmoreus strains separation, select a Hypsizygus marmoreus spore monokaryon bacterial strain as setting out test strain, be numbered T1, match with all sporidium monokaryon bacterial strains; B, if affine with T1 pairing, and clamp connexion appears, then this sporidium monokaryon bacterial strain is all different with A, the B factor of T1 strain mating type gene; If not affine with the T1 pairing, and smooth reaction appears, then this sporidium monokaryon bacterial strain is different with the identical B factor of the A factor of T1 strain mating type gene; If not affine with the T1 pairing, and the fence reaction appears, then this sporidium monokaryon bacterial strain is identical with the T1 strain mating type gene A factor different B factor; If with the no above-mentioned three kinds of phenomenons of T1 pairing, then this sporidium monokaryon strain mating type gene and T1 strains A, the B factor are all identical.
In a step, the Hypsizygus marmoreus strains separation obtains the sporophore that the pairing of sporidium monokaryon bacterial strain comprised the steps: a), hanked ripe parachute-opening Hypsizygus marmoreus, cuts off stem, and lamella simultaneously is flattened on the flat board, places 25 ℃ of incubators to collect spore; B), dip in the sporidium that takes a morsel, in sterilized water, prepare spore suspension, be diluted to that there is 8-12 spore in each visual field under 400 power microscopes with transfering loop; C), get 70 μ l suspension and be coated with flat board, 25 ℃ of dark cultivations 7 days, the spore of having sprouted with the inoculating needle picking at microscopically is together with a fritter substratum, and is transferred on the flat board, cultivates microscopy after 5 days again, determine no clamp connexion after preservation standby; D), two pairings are soaked the cultivation 7～10 days that stands facing each other on the juice substratum for examination monokaryon inoculation in wood chip, temperature remains on 25 ℃, along passing the bacterium piece that two bacterium colonies cut the wide 0.5cm of long 3cm with the perpendicular direction of reaction zone, be transferred on potato dextrose agar (PDA) substratum, under 25 ℃ temperature, continue to cultivate 5～10 days.Wherein, it is to soak in the juice at the 200g wood chip that wood chip soaks the juice substratum, add agar 20g, adding distil water makes to 1000ml again, be transferred to again on the potato dextrose agar (being the PDA substratum) and differentiate, the potato dextrose agar here (being the PDA substratum) is a prior art, directly can purchase on market, has just repeated no more prescription that it is concrete here.
The identification result that is drawn according to the c step is selected affine sporidium monokaryon bacterial strain pairing hybridization, and hybrid strain mycelial growth speed and growth potential and economical character thereof are measured, and carries out the screening of hybrid strain at last.Select affine sporidium monokaryon bacterial strain pairing hybridization to be meant from the sporidium monokaryon bacterial strain of above-mentioned discriminating, to pick out A, carrying out that the B factor is different to match hybridization in twos, under opticmicroscope, observe mycelia and have or not clamp connexion, hybrid strain purifying, the numbering of clamp connexion will be arranged.
Here the employed transfer process that will illustrate especially: the monokaryon bacterial strain of face-off growth was cultivated before this at wood chip and was soaked on the juice substratum, because this substratum nutrition is relatively low, the aerial hyphae of bacterial strain is generally less, and this formation for observe phenomena is favourable; Next will cultivate the bacterium colony of for some time and be transplanted on the PDA substratum, because the nutrition of this substratum is abundanter, original phenomenon is exaggerated at this, shorten 5～10 days than traditional method, and phenomenon is more obvious, simultaneously, is convenient to the picking hybrid strain.And the discrimination method that is adopted in other edible mushroomss in the past all is to carry out on a kind of substratum.So not only will wait bacterium colony almost to cover with flat board and just can differentiate, and phenomenon not clear especially.
Embodiment
The present invention is further described below in conjunction with Fig. 1 and embodiment.
1, parent strain is cultivated, sporidium separates and hybrid strain cultivation PDA substratum: peeling potato 200g, glucose 20g, agar 20g, distilled water 1000ml, pH nature; Culture medium for cultivating: wood chip 40%, cotton seed hulls 24%, wheat bran 35%, gypsum 1%, every bottle of dry weight 240g, it is 60%-62% that the back replenishes water content;
Wood chip soaks the juice substratum: the 200g wood chip soaks juice, adds agar 20g, and adding distil water is to 1000ml.
2, bacterial strain material
Hypsizygus marmoreus SIEF2632 and SIEF3132 are by China Committee for Culture Collection of Microorganisms, and edible mushrooms branch center, Shanghai, agriculture microorganism center provides.
3, the separation of sporidium monokaryon bacterial strain
The sporophore of hanking ripe parachute-opening Hypsizygus marmoreus SIEF2632 and SIEF3132 cuts off stem, and lamella simultaneously is flattened on the flat board, places 25 ℃ of incubators to collect spore; Dip in the sporidium that takes a morsel with transfering loop, in sterilized water, prepare spore suspension, be diluted to that there is 8-12 spore in each visual field under 400 power microscopes; Get 70 μ l suspension and be coated with flat board, 25 ℃ of dark cultivations 7 days, the spore of having sprouted with the inoculating needle picking at microscopically is together with a fritter substratum (about 1mm
2), and be transferred on the flat board, cultivate microscopy after 5 days again, preserve standby behind definite no clamp connexion.
Wherein can produce before two nuclear fissions of clamp connexion (clamp connection) for nucleated mycelium and collude the branched phenomenon of shape (the process synoptic diagram is seen Fig. 2).
3, mating type determines
Spore monocaryon bacterial strain to Hypsizygus marmoreus SIEF2632 and SIEF3132 bacterial strain carries out following experiment respectively: select a Hypsizygus marmoreus spore monokaryon bacterial strain as setting out test strain, be numbered T1, match with all spore monokaryon bacterial strains; Two pairings are soaked face-off cultivation 7～10d on the juice substratum for examination monokaryon inoculation in wood chip, temperature remains on 25 ℃, along passing the bacterium piece that two bacterium colonies cut the wide 0.5cm of long 3cm with the perpendicular direction of reaction zone, be transferred on the PDA substratum, under 25 ℃ temperature, continue to cultivate 5～10d; If affine with the T1 pairing, clamp connexion appears, and then this sporidium monokaryon bacterial strain is all different with A, the B factor of T1 strain mating type gene; If not affine with the T1 pairing, and smooth reaction appears, then this sporidium monokaryon bacterial strain is different with the identical B factor of the A factor of T1 strain mating type gene; If not affine with the T1 pairing, and the fence reaction appears, then this sporidium monokaryon bacterial strain is identical with the T1 strain mating type gene A factor different B factor; If with the no above-mentioned three kinds of phenomenons of T1 pairing, then this sporidium monokaryon strain mating type gene and T1 strains A, the B factor are all identical.
Wherein smooth reaction (flat reaction) is that the mating type A factor of two monokaryon bacterial strains is identical, and B factor difference, its phenomenon are that heterokaryon is very rare, forms depression and the irregular mycelia (Fig. 3) of distortion in old mycelium.Mycelia can be merged, and can not move but examine, and does not have the clamp connexion phenomenon.Fence reaction (barrage reaction) is the mating type A factor difference of two monokaryon bacterial strains, and the B factor is identical, and its phenomenon is that the mycelia of mating bacterium colony repels mutually, is stopping growing near the centre, forms a fence band two bacterium colonies are separated (Fig. 4).Two monokaryon bacterial strains are affine, and the A of mating type gene, the B factor are inequality, and successfully hybridization forms dikaryon, because the dikaryon mycelial growth rate obviously will be faster than monocaryon, colonial morphology as shown in Figure 5.The A of two monokaryon strain mating type genes, the B factor are all the same, and colonial morphology is then as Fig. 6.
4, affine spore monocaryon bacterial strain pairing hybridization
From the above-mentioned Hypsizygus marmoreus SIEF2632 that has differentiated mating type and SIEF3132 monocaryon bacterial strain, pick out A, carrying out that the B factor is different and match hybridization in twos, under opticmicroscope, observe mycelia and have or not clamp connexion, will have the hybrid strain purifying of clamp connexion, numbering to be respectively H1 to H16.
5, hybrid strain mycelial growth rate and growth potential
With the identical cell age hybrid strain mycelia piece of punch tool cut-off footpath 0.8cm, be inoculated in PDA plate culture medium middle part, 25 ℃ of dark 7d that cultivate.Measure colony diameter, the record mycelial growth potential calculates mycelial growth rate, and 5 repetitions are established in each processing.Filter out the bacterial strain H11 of fast growth.
6, the mensuration of hybrid strain experiment in cultivation and economical character
Adopt the cultivation of 850mL Plastic Bottle, every bottled wet feed 600g (water content 60%-62%), 121 ℃ of autoclaving 2h.Put into cooling room after the sterilization, treat that bottle cools off fully, after center material temperature is lower than 25 ℃, use the automatic vaccination machine inoculation, inoculum size is 3% (v/v).Place 24 ℃～25 ℃ of room temperatures then, relative humidity 60%～65%, CO
2Cultivate after 88 days the mycelium stimulation fruiting under concentration 0.02%～0.025% condition.Use ULTRA UM C GR (Computer forMushroom Cultivation) computer environment Controlling System to control fruiting phase temperature (16 ℃), relative humidity (95%-98%) and CO automatically
2Concentration (0.02%～0.03%).
Stretch but edge curls inward (about 8 minutes maturations) time gathers at the sporophore cap, measure the economical character of each bacterial strain, 5 repetitions are established in each processing.Filter out the good bacterial strain H11 of economical character.
7, hybrid strain stability is measured
Stable through 3 its economical characters of experiment in cultivation hybrid strain H11, output is at the 210g/ bottle, than improving 40% about parent's output 150g/ bottle.
The present invention has proposed the discrimination method of hypsizigus marmoreus mating type gene at home first, and be applied to list-single crosses breeding, obtained good effect, illustrate that this method discriminating hypsizigus marmoreus mating type gene is effective, and can significantly reduce the blindness of crossover process, shorten the time of cross-breeding.Simultaneously,, can follow the tracks of crossover process, predict new strain characteristic,, reduce workload, improve efficient for extensive selection cross provides possibility through the discriminating of mating type gene.
Claims (4)
1, a kind of discrimination method of hypsizigus marmoreus mating type gene, it is characterized in that: a, obtain sporidium monokaryon bacterial strain from different Hypsizygus marmoreus strains separation, select a Hypsizygus marmoreus spore monokaryon bacterial strain as setting out test strain, be numbered T1, match with all sporidium monokaryon bacterial strains; B, if affine with T1 pairing, and clamp connexion appears, then this sporidium monokaryon bacterial strain is all different with A, the B factor of T1 strain mating type gene; If not affine with the T1 pairing, and smooth reaction appears, then this sporidium monokaryon bacterial strain is different with the identical B factor of the A factor of T1 strain mating type gene; If not affine with the T1 pairing, and the fence reaction appears, then this sporidium monokaryon bacterial strain is identical with the T1 strain mating type gene A factor different B factor; If with the no above-mentioned three kinds of phenomenons of T1 pairing, then this sporidium monokaryon strain mating type gene and T1 strains A, the B factor are all identical.
2, the discrimination method of a kind of hypsizigus marmoreus mating type gene according to claim 1, it is characterized in that: in a step, the Hypsizygus marmoreus strains separation obtains the sporophore that sporidium monokaryon bacterial strain comprised the steps: a), hanked ripe parachute-opening Hypsizygus marmoreus, cut off stem, lamella simultaneously is flattened on the flat board, places 25 ℃ of incubators to collect spore; B), dip in the sporidium that takes a morsel, in sterilized water, prepare spore suspension, be diluted to that there is 8-12 spore in each visual field under 400 power microscopes with transfering loop; C), get 70 μ l suspension and be coated with flat board, 25 ℃ of dark cultivations 7 days, the spore of having sprouted with the inoculating needle picking at microscopically is together with a fritter substratum, and is transferred on the flat board, cultivates microscopy after 5 days again, determine no clamp connexion after preservation standby; D) two pairings are soaked the cultivation 7～10 days that stands facing each other on the juice substratum for examination monokaryon inoculation in wood chip, temperature remains on 25 ℃, along passing the bacterium piece that two bacterium colonies cut the wide 0.5cm of long 3cm with the perpendicular direction of reaction zone, be transferred on the potato dextrose agar, under 25 ℃ temperature, continue to cultivate 5～10 days.
3, the discrimination method of a kind of hypsizigus marmoreus mating type gene according to claim 1, it is characterized in that: select affine sporidium monokaryon bacterial strain pairing hybridization according to the identification result that the b step is drawn, hybrid strain mycelial growth speed and growth potential and economical character thereof are measured, carried out the screening of hybrid strain at last.
4, the discrimination method of a kind of hypsizigus marmoreus mating type gene according to claim 2 is characterized in that: in the d step, it is to soak in the juice at the 200g wood chip that wood chip soaks the juice substratum, add agar 20g, and adding distil water makes to 1000ml again.
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| CN103389306A (en) * | 2013-07-24 | 2013-11-13 | 上海市农业科学院 | Method for detecting clamp connection in edible fungi mating type research |
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| CN103477993A (en) * | 2013-01-25 | 2014-01-01 | 上海丰科生物科技股份有限公司 | Pure white new hypsizigus marmoreus bacterial strain |
| CN103389306A (en) * | 2013-07-24 | 2013-11-13 | 上海市农业科学院 | Method for detecting clamp connection in edible fungi mating type research |
| CN104673787B (en) * | 2013-12-02 | 2019-05-31 | 上海市农业科学院 | Molecular labeling, detection method and the application of spot jade gill fungus bacterial strain SIEF2632 |
| CN104673787A (en) * | 2013-12-02 | 2015-06-03 | 上海市农业科学院 | Molecular marker, detection method and application of hypsizigus marmoreus strain SIEF2632 |
| CN106416751A (en) * | 2016-09-27 | 2017-02-22 | 四川省农业科学院土壤肥料研究所 | Method for cultivating morchella single-spore strains by adopting 'artificial pollination' |
| CN106416751B (en) * | 2016-09-27 | 2019-11-22 | 四川省农业科学院土壤肥料研究所 | The method that hickory chick single-ascospore strain is cultivated using " artificial pollination " |
| CN111394491A (en) * | 2019-01-03 | 2020-07-10 | 中国科学院微生物研究所 | Mushroom mating type molecular marker and application thereof in identification of mushroom mating type |
| CN110358858A (en) * | 2019-07-29 | 2019-10-22 | 上海市农业科学院 | A kind of method and primer of mating type that identifying spot jade gill fungus White strain monocaryon |
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