CN108642136A - A kind of thermophilic preparation method for belonging to fungi pcr template - Google Patents

Abstract

This application provides a kind of thermophilic preparation methods for belonging to (Thermomyces) fungi pcr template comprising following steps：1) by the mycelium suspended in TE buffer solutions of the thermophilic category fungi, thallus suspension liquid is obtained；2) thallus suspension liquid is heated at least 10 minutes at 90 DEG C or more of temperature, the thallus suspension liquid cracked is used as pcr template.The application obtains the thermophilic method for belonging to fungal gene group and can be used for the thermophilic PCR identifications for belonging to genetic of fungi transformant, biomass that this method uses is small, can shorten the incubation time of transformant and then realize and can be carried out the detecting of transformant, simple and efficient to handle, time saving and energy saving, economic and asepsis environment-protecting in culture early stage, and subsequent PCR high sensitivities, high specificity, it is possibly realized for extensive high flux screening transformant, the method for Rapid identification thermophilic fungal transformant based on this has important scientific meaning and value.

Description

A kind of thermophilic preparation method for belonging to fungi pcr template

Technical field

This application provides a kind of thermophilic preparation methods for belonging to (Thermomyces) fungi pcr template.

Background technology

Thermophilic category fungi (Thermomyces) is a kind of warm Special Fungi monoid suitable for being grown at 45-50 DEG C, point Cloth is extensive, finds there is presence from the torrid zone of the earth, subtropical zone, temperate zone, frigid zone and humid region and arid biogeographic zone.Thermophilic category fungi Because it has a series of unique enzymes and metabolite into the cell so that it under the high temperature conditions there is unique existence to adapt to energy Power.Again because of many of which enzyme and metabolite, such as thermophilic enzyme and bioactive small molecule, have in production and life There is important economic value so that thermophilic fungal becomes current research hot spot.

Genetic transformation is the important means of the biological molecular biology of research.In genetic transformation, most conventional use of side Method is mainly by using the genome of transformant as template, carrying out PCR screenings.Tradition obtains the side of fungal DNA genomic templates Method is all based on greatly CTAB and SDS methods, and both methods is needed a large amount of hyphal cell liquid nitrogen grinding, and not only thalline dosage is big (50mg or more), and it is cumbersome and time-consuming (1-2 hour)；In addition, the phenol used during extraction, chloroform There is equal organic solvents certain toxicity, these factors to have seriously affected experiment progress, is unfavorable for extensive high flux screening and turns Beggar, it is no exception for thermophilic category fungi.

If a kind of thermophilic side for belonging to fungal gene group template of the acquisition small and swift to operate using biomass can be developed Method is possibly realized for extensive high flux screening transformant, and the method for excavating Rapid identification thermophilic fungal transformant has important Scientific meaning and value.

Invention content

One of the application provides a kind of thermophilic preparation method for belonging to (Thermomyces) fungi pcr template comprising with Lower step：

1) by the mycelium suspended in TE buffer solutions of the thermophilic category fungi, thallus suspension liquid is obtained；

2) thallus suspension liquid is heated 8 minutes to 10 minutes at 90 DEG C or more of temperature, the thalline cracked Suspension is used as pcr template.

In a specific embodiment, in step 2), the temperature of heating is 97 to 100 DEG C.

In a specific embodiment, in step 2), the time of heating is 10 minutes.

In a specific embodiment, the method further includes the step 3) after step 2), by the bacterium of the cracking Liquid suspension freezing processing 5 minutes or more is restored preferably by after the thallus suspension liquid freezing processing of the cracking 8 to 15 minutes The thallus suspension liquid of the cracking of freezing processing is obtained to room temperature to use for use as pcr template.Such as the temperature of freezing processing is -20 DEG C or less.

In a specific embodiment, the method further includes step 4), by the thalline of the cracking in step 2) The thallus suspension liquid of the cracking of freezing processing in suspension or step 3) centrifuges, and obtains supernatant as pcr template.

In a specific embodiment, the mass volume ratio of the mycelium and the TE buffer solutions is (0.1mg- 10mg)：100μl.

In a specific embodiment, the TE buffer solutions include the Tris-HCl and 0.05mM of 8mM to 12mM extremely The EDTA of 0.5mM, the TE pH of cushioning fluid are 7 to 8.

In a specific embodiment, the TE buffer solutions include the Tris-HCl and 0.1mM of 10mM to 12mM extremely The EDTA of 0.2mM, the TE pH of cushioning fluid are 7.5 to 7.8.

In a specific embodiment, the thermophilic category fungi is thermophilic Du Pont bacterium (Thermomyces dupontii) And/or thermophilic cotton wool bacterium (Thermomyces lanuginosus).

The two of the application provide a kind of thermophilic identification method for belonging to (Thermomyces) fungi positive genetic transformation, It includes the following steps：With thermophilic category (Thermomyces) as described in the method acquisition as described in any one of one of the application Then the pcr template of fungi carries out PCR screenings.

In a specific embodiment, sequence verification analysis can also be carried out to PCR product.

The advantageous effect of the application：

By the exploration of many experiments of inventor, it is found that the application obtains the thermophilic method for belonging to fungal gene group can be with For the thermophilic PCR identifications for belonging to genetic of fungi transformant, biomass that this method uses is small, when can shorten the culture of thermophilic fungal Between at least 2 to 3 days, and then realize culture early stage can be carried out thermophilic fungal wild type and mutant strain detection, behaviour Make simple and efficient, time saving and energy saving, economic and asepsis environment-protecting, and subsequent PCR high sensitivities, high specificity is extensive high Flux screening transformant is possibly realized, and the method for Rapid identification thermophilic fungal transformant based on this has important science Meaning and value.

Description of the drawings

Fig. 1 is the ER gene knockout schematic diagrames of thermophilic Du Pont bacterium NRRL 2155.

The mutant strain PCR detection figures that Fig. 2 is the thermophilic Du Pont bacterium NRRL 2155 for knocking out ER genes.Wherein, M is The Marker of 2000bp is followed successively by 2,000bp, 1,000bp, 750bp, 500bp, 250bp and 100bp from top to bottom；It is negative Control is to be obtained using extracting the genomic DNA of thermophilic 2155 wild strains of Du Pont bacterium NRRL using CTAB methods as template progress PCR Product is obtained, WT is labeled as.

The PCR detection figures that Fig. 3 is embodiment 3-12, the template of all PCR are taken from the mycelia after culture 3d.Wherein The stripe size of Marker (M i.e. in figure) is followed successively by 5,000bp, 3,000bp, 2,000bp, 1,500bp, 1 from top to bottom, 000bp、750bp、500bp、250bp、100bp。

Fig. 4 is the PCR detection figures of embodiment 13, and the stripe size of wherein Marker (M i.e. in figure) is from top to bottom successively For 5,000bp, 3,000bp, 2,000bp, 1,500bp, 1,000bp, 750bp, 500bp, 250bp, 100bp.

Fig. 5 is the PCR detection figures of comparative example 1 and 2.Swimming lane 1 is blank control；Swimming lane 2-4 be followed successively by comparative example 1 with The DNA of the hyphae length extraction of 0.1mg, 2mg, 10mg makees the PCR results of template progress；Swimming lane 5-7 is followed successively by comparative example 2 and is cultivating Make the PCR results of template progress after 5d, 6d and 7d with the hyphae length of 10mg extraction DNA.The item of wherein Marker (M i.e. in figure) Band size be followed successively by from top to bottom 5,000bp, 3,000bp, 2,000bp, 1,500bp, 1,000bp, 750bp, 500bp, 250bp、100bp。

Fig. 6 is the carry out ITS segments PCR that comparative example 3 handles that brewing yeast cell makees template with the application method and CTAB Detection figure.Swimming lane 1 be using the application method treated PCR that sample carries out as template as a result, swimming lane 2 is CTAB methods extract after Sample be template carry out PCR as a result, swimming lane 3 be by template of the plasmid containing ITS segments carry out PCR results.Wherein The stripe size of Marker be followed successively by from top to bottom 5,000bp, 3,000bp, 2,000bp, 1,500bp, 1,000bp, 750bp, 500bp、250bp、100bp。

Specific implementation mode

The application is described further with reference to embodiment, but the exemplary only explanation of the embodiment of the present application, it should No matter embodiment does not constitute the restriction to the application under any circumstance.

Reagent used in this application can routine business acquisition.

Thermophilic category fungi is thermophilic, and Du Pont bacterium hermomyces dupontii NRRL 2155 are commercially available, thermophilic cotton wool Bacterium Thermomyces lanuginosus YM3-4 can be obtained from Yunnan protection and utilization of the biological resources National Key Laboratory, S. cervisiae Saccharomyces cerevisiae AH109 are commercially available.

P buffer solutions：MgSO₄·7H₂O 39.435g, sodium citrate 11.764g adjust pH after 150mL distillation water dissolutions are added Value=5.5, is settled to 200mL.Conventional high-pressure sterilizes.

N buffer solutions：Sorbierite 9.105g, sodium citrate 0.735g, be added 30mL distilled water, after dissolving, be adjusted to pH value= 5.8, it is settled to 50mL.Conventional high-pressure sterilizes.

STC solution：Sorbierite 18.21g, CaCl₂·2H₂O 0.735g are added 60mL distilled water, 1M are added after dissolving Tris-HCl buffer solutions (pH=8.0) 5mL, is then settled to 100mL.Conventional high-pressure sterilizes.

60%PEG4000 solution：12g PEG4000 are dissolved by heating in 10mL distilled water, and distilled water constant volume is then added To 20mL.Conventional high-pressure sterilizes.

KTC solution：6.710g KCl are dissolved in 30mL distilled water, and 7.5mL 1M Tris-HCl buffer solutions are then added (pH is adjusted to 8.0)；1.104g CaCl₂·2H₂O is dissolved in 4mL distilled water, then by CaCl₂·2H₂O solution pours into KCl+1M In Tris-HCl (pH is adjusted to 8.0) solution, it is eventually adding distilled water and is settled to 50mL.Conventional high-pressure sterilizes.

PTC conversion fluids：Sterile 60%PEG4000 solution and sterile KTC solution are with volume ratio 2：1 mixing.

The KCl that terminate liquid can be 0.6M is digested, i.e. 2.684g KCl are dissolved in 40mL distilled water, constant volume to 60mL. Conventional high-pressure sterilizes.

PDA solid mediums：Peeled potatoes 200g cuts fritter and boils 20min to 30min, four layers of filtered through gauze, grape Sugared 20g, agar 18-20g are settled to 1L, natural pH.TB3 fluid nutrient mediums：Sucrose 200g, yeast extract 3g, caseinhydrolysate 3g is settled to 1L with distilled water.Solid medium then needs to add 0.75% agar powder.Conventional high-pressure sterilizes.

YPS fluid nutrient mediums：Yeast extract 4g, soluble starch 1.5g, KH₂PO₄1g, MgSO₄·7H₂O 0.5g, it is molten Add distilled water constant volume to 1L after solution.Conventional high-pressure sterilizes.

TB3 culture mediums：Sucrose 200g, yeast extract 3g, caseinhydrolysate 3g are settled to 1L with distilled water.Solid culture Base then needs to add 0.75% agar powder.Conventional high-pressure sterilizes.

TYGA solid mediums：Yeast extract 5g, peptone 10g, molasses 5g, glucose 10g, agar 18-20g add Water is settled to 1L.Conventional high-pressure sterilizes.

Embodiment 1

Knock out the structure of plasmid

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated 7 days in case, then scrape about 100mg mycelia, Genomic PCR template is extracted using CTAB methods, steps are as follows：

(1) 10mL CTAB dissociating buffers (CTAB 4g/L, NaCl 16.364g/L, 1M Tris-HCl 20ml) is added Enter in 50mL centrifuge tubes, is placed in 60 DEG C of water-baths and preheats.

(2) mycelia for weighing 100mg mycelia, is placed in the mortar of precooling, pours into liquid nitrogen, as early as possible grinds mycelia.

(3) abrasive flour is taken to be directly added into the CTAB dissociating buffers of preheating, gently rotation is allowed to mixing.

(4) sample keeps the temperature 30 minutes in 65 DEG C.

(5) chloroform-isoamyl alcohol for adding isometric (10mL), gently overturns mixing.

(6) 4000rpm is centrifuged 10 minutes at room temperature.

(7) upper strata aqueous phase is sucked in another clean centrifuge tube with an opening larger dropper, the different of 2/3 volume is added Propyl alcohol, gently mixing, makes nucleic acid precipitate.

(8) DNA is collected with following methods：

If being in visible filiform DNA, it can be stirred with glass bar, be transferred to 10-20mL washing buffers (75% alcohol) In.If DNA is in cloud, it can be centrifuged 1-2 minutes in 2000rpm, carefully outwell supernatant, added in loose precipitation 10-20mL washing buffers, gently rotating centrifuge tube makes nucleic acid precipitation suspend.

(9) it is washed at least twice with 75% ethyl alcohol.

(10) nucleic acid is collected again according to (8) method, and according to (9) method secondary washing.

(11) it draws precipitation with glass bar or 2000rpm is centrifuged 10 minutes.Supernatant carefully is removed, makes DNA at room temperature Precipitate drying.

(12) in DNA precipitations being dissolved in 1 to 1.5mL TE, DNA solution is obtained, is saved backup for -20 DEG C after packing.

ER genes (EM_EST is obtained with the method for PCR:EB064217 upstream and downstream homology arm segment), wherein with ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2) is that primer obtains ER upstream region of gene homology arm segment ER (up), with ER3F (SEQ ID No.3)/ER3R (SEQ ID No.4) is that primer obtains ER downstream of gene homology arm segment ER (down)；With plasmid PAg1-H3 (receive and taught in Texas ,Usa medical research institute An Zhiqiang) is template, with primer HygB-F (SEQ ID No.5) and HygB-R (SEQ ID No.6) progress PCR acquisition hygromycin resistance expression segments (Hyg).It is with pUC19 plasmids Skeleton carrier presses ER (up), Hyg, ER (down) these three segments with In-Fusion HD Cloning seamless link technologies It, then will be even according to being sequentially connected to after Spe I and Hind III digestions in pUC19 linear plasmid segments from 5 ' ends to 3 ' ends Object of practicing midwifery extremely conversion bacillus coli DH 5 alpha competence, picking transformant, with ER5F (SEQ ID No.1) and ER3R (SEQ ID No.4 it is) that primer carries out PCR verifications and identification positive transformant is sequenced, as obtains pUC19--ER (up)-Hyg-ER (down) Knockout plasmid.Using the knockout plasmid as template, with ER5F (SEQ ID No.1) and ER3R (SEQ ID No.4) be primer into Row PCR amplification, which obtains, knocks out full length fragment ER (up)-Hyg-ER (down) (SEQ ID No.7,6940bp).

Embodiment 2

Preparation, conversion and the verification of thermophilic 2155 protoplasts of Du Pont bacterium NRRL

The preparation of TE buffer solutions：10mM Tris-HCl and 0.1mM EDTA, pH 7.5.

(1) it takes the inoculated by hypha block of thermophilic Du Pont bacterium NRRL 2155 at PDA culture medium center, is positioned over 45 DEG C of cultures It is cultivated 7 days in case.Culture is shaved from above-mentioned tablet with 1mL pipette tips and sterile water (polysorbas20 for adding 0.05%), with four Layer lens wiping paper filtering, by the liquid subpackage containing spore to 1.5mL centrifuge tubes, 10000rpm, room temperature 5min centrifugal enrichment spores Son abandons supernatant, enrichment to 1 × 10⁸After a/mL twice with sterile water wash.

(2) it draws 200 μ L spore suspensions to be transferred in 100mL YPS fluid nutrient mediums, 45 DEG C, 180rpm shake cultures 20h。

(3) mycelia that step (2) is cultivated is poured into sterile funnel (including four layers of lens wiping paper) and mycelium is collected by filtration.

(4) mycelium is washed twice with P buffer solns.

(5) the appropriate mycelia of picking is transferred in the sterile triangular flask of 100mL, and cell walls of the 20mL through filtration sterilization is added and splits Solve liquid (3mL contains the N buffer solution+17mL P buffer solutions of 0.3g/ml yeast wall breaking enzyme (Lysing enzymes)).

5.5h is digested in (6) 28 DEG C of 90rpm shaking tables, until mycelia is complete by all cracking.

(7) mycelia relic is filtered with sterile funnel (including six layers of lens wiping paper), collects and digests complete protoplast, be added The KCl enzymolysis reactions of 20mL 0.6M, obtain lysate solution.

(8) lysate solution is dispensed into 1.5mL centrifuge tubes, 5000rpm, 4 DEG C of centrifugation 10min.

(9) supernatant is slowly outwelled, every 6 pipe to 10 pipe protoplasts (lysate) is enriched with to a pipe, 1mL is then added STC solution, 5000rpm, 4 DEG C of centrifugation 10min.

(10) supernatant is slowly outwelled, often 1mL STC solution, 5000rpm is added in pipe, and it is molten that STC is added in 4 DEG C of centrifugation 10min Protoplast is adjusted to 1 × 10 by liquid⁸The final concentration of a/mL.

(11) protoplast prepared is dispensed into the sterile EP tube of 2mL, often 200 μ L of pipe, is placed in for use on ice.

(12) the knockout full length fragment that recycling is purified in embodiment 1 (SEQ ID No.7) 10 μ g are added to above-mentioned acquisition Final concentration of 1 × 10⁸In the Protoplast suspension of a/mL, soft mixing is rapid to place 40min on ice.

(13) 1mL PTC liquid is added into the solution after ice bath, is positioned in 45 DEG C of incubators and stands 30min, then It is positioned over 10min on ice.

(14) it draws above-mentioned mixing liquid and is spread evenly across TB3 solid culture primary surfaces, 150 μ L are coated with per ware, are positioned over 45 16h is cultivated in DEG C incubator.

(15) after 16h, the planar surface that protoplast mixed liquor is coated in previous step covers last layer and contains 200 μ g/ The TB3 culture mediums of mL hygromycin Bs.

(16) after growing transformant on solid plate, choose a certain number of transformants be individually transferred to respectively containing On the TYGA solid plates of 200 μ g/mL hygromycin Bs；Transformant is on the TYGA solid plates that Hygromycin B concen is 200 μ g/mL Secondary culture 3 days.

(17) pcr template is prepared as follows：In step (16) on culture plate, the mycelium of scraping about 1mg is equipped with In the 1.5ml EP pipes of 100 μ L TE buffer solutions, lid is covered tightly, vortex oscillation 10s makes mycelia mix well in TE buffer solutions, Thallus suspension liquid is obtained, by the 1.5ml EP pipe water bath processings equipped with thallus suspension liquid, that is, is placed in boiling water boiling in water-bath 10min is then cooled to room temperature, the thallus suspension liquid cracked, as pcr template.

(18) PCR verification operations are as follows：

By 1 μ l, TaqTM enzyme of thallus suspension liquid (or referred to as DNA solution) (the TAKARA public affairs for the cracking that above-mentioned (17) obtain Department, article No.：R001A), sense primer ERF2 (SEQ ID No.8)；Downstream primer ERR2 (SEQ ID No.9).PCR programs are： 98 DEG C of 2min, 58 DEG C of 30s, 72 DEG C of 3min, 30 cycles；72℃5min；12℃10s.Ago-Gel electricity is carried out to PCR product Swimming identification.

As a result see Fig. 2, (swimming lane 1-7) can effectively amplify purpose band in 7 transformants of detection, and explanation is used for The genomic templates of PCR are qualified.Wherein, according to amplified band as a result, the i.e. item of the size of amplified band and amplified band Number may determine that the corresponding transformant of swimming lane 3 to 5 is the positive transformant knocked out completely.

Embodiment 3

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated 5 days in case.

Pcr template is prepared with the step (17) in embodiment 2.The difference is that the mycelia selected is the present embodiment culture The mycelia of obtained wild mushroom；And the formula of TE buffer solutions is：8mM Tris-HCl and 0.05mM EDTA, pH 7.

PCR verification operations are the same as the step (18) in embodiment 2.The difference is that the primer that PCR is used is ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2), pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 3 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 4

Pcr template is prepared with embodiment 3.It the difference is that only the formula of TE buffer solutions, i.e.,：10mM Tris-HCl and 0.2mM EDTA, pH 7.8.

PCR verification operations are the same as embodiment 3.It the difference is that only that the template that PCR is used is what the present embodiment was prepared Template.

As a result see the swimming lane 4 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 5

Pcr template is prepared with embodiment 3.It the difference is that only the formula of TE buffer solutions, i.e.,：12mM Tris-HCl and 0.5mM EDTA, pH 8.0.

PCR verification operations are the same as embodiment 3.It the difference is that only that the template that PCR is used is what the present embodiment was prepared Template.

As a result see the swimming lane 5 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 6

Pcr template is prepared with embodiment 3.It the difference is that only the formula of TE buffer solutions, i.e.,：15mM Tris-HCl and 0.1mM EDTA, pH 7.5.

PCR verification operations are the same as embodiment 3.It the difference is that only that the template that PCR is used is what the present embodiment was prepared Template.

As a result see the swimming lane 6 in Fig. 3, may determine that from figure cannot amplify band, illustrate Tris-HCl excessive concentrations The Genomic PCR template of the fungi cannot effectively be obtained.

Embodiment 7

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated 5 days in case.

Pcr template is prepared with the step (17) in embodiment 2.The difference is that the mycelia selected is the present embodiment culture The mycelia of obtained wild mushroom；And the temperature of the water bath processing of thallus suspension liquid is 90 DEG C.

PCR verification operations are the same as the step (18) in embodiment 2.The difference is that the primer that PCR is used is ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2), pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 7 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 8

Pcr template is prepared with embodiment 7.The difference is that the temperature of the water bath processing of thallus suspension liquid is 97 DEG C.

PCR verification operations are the same as embodiment 7.The difference is that pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 8 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 9

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated 5 days in case.

Pcr template is prepared with the step (17) in embodiment 2.The difference is that the mycelia selected is the present embodiment culture The mycelia of obtained wild mushroom；And the time of the water bath processing of thallus suspension liquid is 8min.

PCR verification steps are the same as the step (18) in embodiment 2.The difference is that the primer that PCR is used is ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2), pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 9 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 10

Pcr template is prepared with embodiment 9.The difference is that the time of the water bath processing of thallus suspension liquid is 15min.

PCR verification operations are the same as embodiment 9.The difference is that pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 10 in Fig. 3, may determine that from figure cannot amplify band, illustrate due to water bath time mistake Long, the thallus suspension liquid of the cracking obtained under the conditions of the present embodiment is not suitable anymore for as the template of PCR.

Embodiment 11

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated 5 days in case.

Pcr template is prepared with the step (17) in embodiment 2.The difference is that the mycelia selected is the present embodiment culture The mycelia of obtained wild mushroom, and the dosage of mycelia about 0.1mg.

PCR verification steps are the same as the step (18) in embodiment 2.The difference is that the primer that PCR is used is ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2), pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 11 in Fig. 3, may determine that from figure can effectively amplify clearly purpose band, but purpose item Also contain fuzzy non-specific band with lower section.Therefore, although the thalline of the cracking obtained under the conditions of the present embodiment suspends Liquid quality is not template that is best, but may be used as PCR.

Embodiment 12

Pcr template is prepared with embodiment 11.The difference is that the dosage of mycelia about 10mg.

PCR verification operations are the same as embodiment 11.The difference is that pcr template is the template that the present embodiment is prepared.

As a result see the swimming lane 12 in Fig. 3, may determine that from figure can effectively amplify purpose band, and without non-specific item Band, and compared with the result in embodiment 2, PCR product band is more bright, shows the cracking obtained under the implementation condition Thallus suspension liquid (i.e. pcr template) is more excellent.

Embodiment 13

By the inoculated by hypha block of thermophilic cotton wool bacterium (Thermomyces lanuginosus) YM3-4 in PDA plate, place It is cultivated 5 days in 45 DEG C of incubators.

The mycelium for scraping about 10mg is packed into equipped with 100 μ L TE buffer solutions (10mM Tris-HCl and 0.1mM EDTA, pH 7.5) in 1.5ml EP pipes, lid is covered tightly, vortex oscillation 10s makes mycelia mix well in TE buffer solutions, and it is outstanding to obtain thalline 1.5ml EP pipe water bath processings equipped with thallus suspension liquid are placed in boiling water boiling 10min in water-bath, then cooled down by supernatant liquid To room temperature, the thallus suspension liquid that is cracked.

With primer I TS1 (SEQ ID No.10) and ITS4 (SEQ ID No.11) and the thallus suspension liquid of cracking to open country The raw thermophilic cotton wool bacterium of type carries out the PCR amplification of ITS segments.

As a result see Fig. 4, may determine that from figure can Successful amplification go out the ITS segments of the fungi, illustrate in the present embodiment Under the conditions of the thallus suspension liquid of cracking that obtains may be used as the template of PCR.

Comparative example 1

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated 7 days in case.

Genomic DNA is extracted by the method for CTAB.Referring to (1) the step of embodiment 1 to (12).Wherein, it takes respectively The mycelia of 0.1mg, 2mg, 10mg, 50mg and 100mg carries genome.

With ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2) for primer, with the present embodiment in 5 kinds of different hyphae lengths The genomic DNA extracted under (0.1mg, 2mg, 10mg, 50mg and 100mg) is that template carries out PCR verifications.

As a result Fig. 3 and Fig. 5 are seen, from figure 5 it can be seen that cannot be to mesh when hyphae length is 0.1mg, 1mg and 10mg Standard film section is effectively expanded, and when illustrating that CTAB methods is used to extract genomic DNA as pcr template, hyphae length is in 0.1- Genome amplification can not be effectively obtained between 10mg；And from figure 3, it can be seen that when hyphae length reaches 50mg or more, it uses CTAB methods extract genomic DNA as pcr template, and effective amplification to target fragment may be implemented.Illustrate to use in mycelium Amount 10mg and it is following when, using CTAB methods extraction genomic DNA cannot function as the template for PCR amplification.

Comparative example 2

Thermophilic 2155 wild mushrooms of Du Pont bacterium NRRL are inoculated by slant medium in PDA culture medium, 45 DEG C of trainings are positioned over It supports and is cultivated in case.

Respectively when incubation time is 5d, 6d and 7d, 10mg mycelia is taken to carry out the extraction of genomic DNA, other extractions behaviour Make with (1) the step of embodiment 1 to (12).

With ER5F (SEQ ID No.1)/ER5R (SEQ ID No.2) for primer, with the genomic DNA of the present embodiment extraction PCR verifications are carried out for template.

As a result see Fig. 5, may determine that from figure cannot expand to target fragment, illustrate that extracting genomic DNA cannot function as Template for PCR amplification.

Comparative example 3

28 DEG C in 2ml LB liquid mediums, after 220rpm cultures 2d, 5000rpm centrifugations obtain wild-type yeast AH109 Thalline is taken, genomic DNA is extracted with yeast thalline 100mg.

Two kinds of extracting methods are respectively adopted, one kind is CTAB methods, another is the same as step (17) in embodiment 2.

With ITS1 (SEQ ID No.10) and ITS4 (SEQ ID No.11) for primer, respectively obtain in two ways respectively Genomic DNA be template amplification ITS segments carry out PCR verifications.

As a result as can be seen from Figure 6, the genomic DNA obtained using the method for 2 step of embodiment (17) cannot effectively expand ferment The target fragments of female bacterium, and traditional CTAB methods then can effectively expand the target fragments of saccharomycete.As it can be seen that the present processes The pcr template of the fungies such as yeast is not suitable for preparing.

Although the application is described with reference to specific implementation mode, it should be appreciated by those skilled in the art In the case of no real spirit and scope for being detached from the application, the various changes that can carry out.Furthermore, it is possible to this Shen Main body, spirit and scope please are variously changed to adapt to specific situation, material, material compositions and method.All These changes are included in the range of claims hereof.

Sequence table

<110>Yunnan University

<120>A kind of thermophilic preparation method for belonging to fungi pcr template

<130> LHA1860307

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213>Artificial sequence (non)

<400> 1

cgccgcttgc ctgtatgacg tag 23

<210> 2

<211> 26

<212> DNA

<213>Artificial sequence (non)

<400> 2

gggcccgggg tgcagccgga cagatc 26

<210> 3

<211> 27

<212> DNA

<213>Artificial sequence (non)

<400> 3

actagtacgg cgattcgaga actggag 27

<210> 4

<211> 27

<212> DNA

<213>Artificial sequence (non)

<400> 4

aagcttgccc gggtaatgcc ttggatc 27

<210> 5

<211> 34

<212> DNA

<213>Artificial sequence (non)

<400> 5

tacgccggac gcatcacggc gattcgagaa ctgg 34

<210> 6

<211> 35

<212> DNA

<213>Artificial sequence (non)

<400> 6

atcatcttct gtcgaggggt gcagccggac agatc 35

<210> 7

<211> 6940

<212> DNA

<213>Artificial sequence (non)

<400> 7

cgccgcttgc ctgtatgacg tagtactctt gtttgatgcc tcattctcgt actgctggaa 60

gtacgcattt cacgacttca gacggtactt gtagagcaca tatgtaccta ggtgactaca 120

gtacctgcct gtaggtacct gtacccaggt agttgcgtag aagtacagtt atcctgatat 180

ttatctgtgg cctgccagaa tggtgtctga atgtacgggg tacaggcaaa gtagtaaaaa 240

tcctgttgac ttacggagta ctccgtatat gacgctgata ctacatcggc agtgccaaaa 300

tgtgtgcggt atgcagttac ctaatggtta cctgtccatc aaagttaacg agtttatcgt 360

agttacagag taactcaatt cagttacata cataggtagt taagttaaat agttaagcat 420

aaaactttac gtaaactgtg gcttgcagcg ttagtggcgg cgcctaaagg atctttccgt 480

tgatcagagg atcagaccct gcaggacctc gtataactta aactcaaccg tcaggttcca 540

tgtggaccgg gccgaaccgg agaccctctg ggtccggaaa tgtaactgcc gacagccgtt 600

gcacaccatt tccgtcagta gtacggagta ctccgtatac ctaggtaggt gctttctctg 660

caactcggcc ttgaggtgta ccagatgaga tttgcagtcg ttggtgattc cggaccagac 720

aaagtcaaac gtgcatactg cctctccatt tccattacaa gcacaatccc atcaccatga 780

tgacctggga gaaccgcgac gtgaaggaca accacctctt gaatttctcc ggtgatcaga 840

ccctgcagga tctcgtatgg catcgtcagg ttcgatctgg accgatccga gccggagacc 900

ctctggggcc ggaaatgtcg ttgctcacgc tctttgcacg agatttccga cggtggtagg 960

atacatgctg cacaattccc ccgtgggcca aatgttatat aggcagtcat tggtgggctt 1020

cggaccagac gaaatcagac cttgaactta cttctccatt tccctttaca agcacaagat 1080

caccatcatg acgacctggg agaaccgaga cgtgacgaac aaccacatcc taccctacgt 1140

ggactcgtcg acggcgccag acgatgtccg cgccgctctc aagacgcttc ccttcgagcg 1200

caacatcttc aaggtgtgat atggaaccca attcgcattc gacgcacgga ccagagagag 1260

ggagagcctc ctactgacca tgataacagc tcctggcgaa tgcgccgacc ttctttccca 1320

ccttcatgaa gctcttgacc tgtgcctggt ctcccaaccg cagtctccgc tcgaccgact 1380

ggcagctggt ggtcctgcgc acggcgtctc tactagacgc cccctacgag tgggatgtca 1440

acgagcccgt ggcccgcgtg tttggcttta ccgatgcgca cctggcatgt ctgcgctccg 1500

gagacctctc gagcaccgag ctgttcaccg atcgccagcg actgatcagt gccatcgtgg 1560

aggagctgtg tctgaagaac cgggttaaca ctgaactcgt tgagaaggcc aaggcggtcc 1620

tgggggtaga ggccctcatg gatttgttct atacccaagg catctacgcc ttcctggcgc 1680

gcaccatgaa cagctgcctg attgattttg acccgcccat cccgggtctg gaggagcagc 1740

tgcggaagta caatgctgcg attattgaga aggagagcca gtataccgat tgatgtgtat 1800

gtagaggatg taacgaatga ctatccttgg ccatgttccg gacgcttgag cgttgtatct 1860

gacagatgag gatgtatatc gtgcttcgga tatgttatag caagatatgg cacagtccct 1920

cagagtactc cgtgttgagt tttggcaggt tatgacacat ctggcgagta cctggggtga 1980

taagaccagt ggtcctgacc aacagactga tgtcagatgc ttgattccac tctatgtcac 2040

agagggtagg gaacgacccc tacttatgta catgggtagg tagatgaggc agaaaaaaca 2100

tggacaaatg aatgtcaggc agacacaaca aacacaagat aataatgaag gacacttgat 2160

taatgtacgc gattatcaga gcattcaaca ctctggacat tctcatatgg aacgcttcca 2220

acagaattct ccagaccgcc accctgtaca ccggaatata cataaacacc agaaagaatg 2280

atagactaca tgctatccaa tcccaagccc tgtgcatcag atctccttag cgactacatc 2340

ctagctactt catcagaatt gttgctacct caagactcaa attgaaagac gggcttgaga 2400

ttccccacca cccctgcaga actctccttg atctgagata gagcctcccg gactcgtttc 2460

tccagcgaac cgtctccttt ccatttaatg atcttcaccg gaggcgcaac cagctgtccc 2520

tgctcaaaga gctccccgaa ccaggtcagg agctggatct gcgcctcctc tgtgtaccgt 2580

gccagggcct gagacagccg gaagttgcga aaggtgattt gcttccagaa gagcagctct 2640

tgcgtcagaa tgatctgtcc ggctgcaccc ctcgacagaa gatgatattg aaggagcact 2700

ttttgggctt ggctggagct agtggaggtc aacaatgaat gcctattttg gtttagtcgt 2760

ccaggcggtg agcacaaaat ttgtgtcgtt tgacaagatg gttcatttag gcaactggtc 2820

agatcagccc cacttgtagc agtagcggcg gcgctcgaag tgtgactctt attagcagac 2880

aggaacgagg acattattat catctgctgc ttggtgcacg ataacttggt gcgtttgtca 2940

agcaaggtaa gtgaacgacc cggtcatacc ttcttaagtt cgcccttcct ccctttattt 3000

cagattcaat ctgacttacc tattctaccc aagcatcgat atgaaaaagc ctgaactcac 3060

cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 3120

gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt 3180

cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt 3240

tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct 3300

gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga 3360

actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct 3420

tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg 3480

gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga 3540

cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga 3600

ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga 3660

caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata 3720

cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg 3780

ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct 3840

ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc 3900

ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac 3960

acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga 4020

tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagagta gatgccgacc 4080

gggatccact taacgttact gaaatcatca aacagcttga cgaatctgga tataagatcg 4140

ttggtgtcga tgtcagctcc ggagttgaga caaatggtgt tcaggatctc gataagatac 4200

gttcatttgt ccaagcagca aagagtgcct tctagtgatt taatagctcc atgtcaacaa 4260

gaataaaacg cgtttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg 4320

cattggacct cgcaacccta gtacgccctt caggctccgg cgaagcagaa gaatagctta 4380

gcagagtcta ttttcatttt cgggagacga gatcaagcag atcaacggtc gtcaagagac 4440

ctacgagact gaggaatccg ctcttggctc cacgcgacta tatatttgtc tctaattgta 4500

ctttgacatg ctcctcttct ttactctgat agcttgacta tgaaaattcc gtcaccagcc 4560

cctgggttcg caaagataat tgcactgttt cttccttgaa ctctcaagcc tacaggacac 4620

acattcatcg taggtataaa cctcgaaaat cattcctact aagatgggta tacaatagta 4680

accatggttg cctagtgaat gctccgtaac acccaatacg ccggccgaaa cttttttaca 4740

actctcctat gagtcgttta cccagaatgc acaggtacac ttgtttagag gtaatccttc 4800

tttctagagg atcctctacg ccggacgcat cacggcgatt cgagaactgg agttcccaat 4860

gaacacccgt gactcagtcc gccgtacttc atatttataa tcacgacacg cattgcttgc 4920

gttgaatgga acttggaagc atcgctgttg cggacactgt gagaagtacc taggtaccac 4980

aagggacatt tccgaaggcc ccacagcatt tccgggcgag cagcgccgct cagaataatc 5040

atacccagca aaacaccgga agagctacga aacatcagga ttgaaagcac ggaaatggtc 5100

tgatgtatta ttaatgaccg tttgacgaag tatggagccc acagcatggc tcgtggcata 5160

cataaggccc ggacggtcga cgggcaacaa acggatgcag acgccgttgg gagccatcat 5220

ccctcgagat agagactggg cacaatgtct gcaaaggtgc tagaagacac tccttcgcct 5280

tcgtccgagc cgccctcgga gacgtccgct gagaagcctt cgtcaccacg tgatgttcat 5340

ggcatcaagg tcagttccat cccttttccc cagcgtgtgc cctaccaatc actaacatac 5400

gggacagtgg gtgcttgtcg tggtctcgct gctctcgtcc ctctttctct acgccctgga 5460

caataccgtc gttgccaatg tccagcccaa ggttgtccta accttcggcc atgtcgcgtt 5520

ggttccctgg ctcggcgtct cctttgccct agcctcggcc gccacgaccc tgatctggtc 5580

caaagcgtac ggaaccttct ccgcgaagaa gctgtacatt ggtagcactg ccgtattcat 5640

ggctggctcg gccctgtgcg gtgccgcccc agacatcgac agcttcatcg tcggccgcgc 5700

cattgcagga gccgccgggt gcggcatgta catgggtctg ttgacccttc tttccgtcac 5760

gactgacaac atcgagcggc ccaagtacct cagtctcacg ggcttgattt ggggtattgg 5820

tacggtgctc gggcccgtcg tcggcggcgc gttcggtgac agcagcgcca cctggcgttg 5880

ggcgttttac ctgaatctgc tggtcggcgt ggtggtcgcc ccgatttatt tccttctcct 5940

gcctgatttc cagccacaga agggcgagcc gctcctggcg cgtctggcac aaatcgacta 6000

cctcggcggc ctgctgtcca ttggcggtct gctgtgtctg atcatggcca tgaactttgg 6060

cggcgtgctg tggccgtgga accaccgcaa cagcattggt ctgttcgtgg gggctggcgt 6120

gcttatcatc ctcttcgtcg ttcagcaggt cttactcatc ggcacgtcat ttaactcgcg 6180

catgttccca atgcatttct ggcgcagccg ctcgttgatt gtgctgttcc tcactatgag 6240

tgagtttcca actcgacatc tgtaaccctg cgatcatccg ctgactttcg cagtgctggc 6300

aacgtttggc agctttatcg gcatctacta cctgcccatc tactaccagt tcgcacgcgg 6360

cagcaccgcc atggagacct ccgttcattt gctccccttc attctgttcc tggtggcctt 6420

caacctggct aacggccagt tcatggccaa gacgggctac tactacccgt ggtacatagt 6480

cgggggcgcg ttggaactca tcggcggcgt tttaatgtgt aagtatccca tcctcttccc 6540

tccccatccc cttttggcag acgagcaatc cgctgacaaa cgcagacttt gtcgacgaga 6600

acacaagcga tgccaaaatc tatgggtaca caatcatcct tggcacgggc gtgggctgtt 6660

tctgccaggc gggctttgcc gtggcgcaga tgaaagtgaa ggcttccgag atcccgtact 6720

gcgtgggttt catgaccgtt ggccagatgt tgggaattgt gcttggcacg ggcatcgcgg 6780

gtgctctgtt cgtcaactcg gcccaggatt ccctccgcgc tgttttccct gatgccagcg 6840

aggaggagat ctccaatgcc atttccggcg tcggcagcga gctgctgact tctgccgggc 6900

cagctaagga gcaggccggg atccaaggca ttacccgggc 6940

<210> 8

<211> 19

<212> DNA

<213>Artificial sequence (non)

<400> 8

gcagtcggtc ccaattccc 19

<210> 9

<211> 21

<212> DNA

<213>Artificial sequence (non)

<400> 9

atgcctaatc cggtcaactt g 21

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence (non)

<400> 10

ccgtaggtgt gaacctgcgg 20

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence (non)

<400> 11

tcctccgctt attgatatgc 20

Claims (10)

1. a kind of thermophilic preparation method for belonging to (Thermomyces) fungi pcr template comprising following steps：

1) by the mycelium suspended in TE buffer solutions of the thermophilic category fungi, thallus suspension liquid is obtained；

2) thallus suspension liquid is heated 8 minutes to 10 minutes at 90 DEG C or more of temperature, the thalline cracked suspends Liquid is used as pcr template.

2. according to the method described in claim 1, it is characterized in that, in step 2), the temperature of heating is 97 to 100 DEG C.

3. method according to claim 1 or 2, which is characterized in that in step 2), the time of heating is 10 minutes.

4. method as claimed in any of claims 1 to 3, which is characterized in that the method further include step 2) it Step 3) afterwards, by the thallus suspension liquid freezing processing 5 minutes or more of the cracking, preferably by the thallus suspension liquid of the cracking After freezing processing 8 to 15 minutes, restore the thallus suspension liquid for the cracking for obtaining freezing processing to room temperature makes for use as pcr template With.

5. method as claimed in any of claims 1 to 4, which is characterized in that the method further includes step 4), will The thallus suspension liquid of the cracking in step 2) or the centrifugation of the thallus suspension liquid of the cracking of the freezing processing in step 3), obtain Supernatant is as pcr template.

6. method as claimed in any of claims 1 to 5, which is characterized in that the mycelium is buffered with the TE The mass volume ratio of liquid is (0.1mg-10mg)：100μl.

7. method as claimed in any of claims 1 to 6, which is characterized in that the TE buffer solutions include 8mM extremely The EDTA of the Tris-HCl and 0.05mM to 0.5mM of 12mM, the TE pH of cushioning fluid are 7 to 8.

8. the method according to the description of claim 7 is characterized in that the TE buffer solutions include the Tris-HCl of 10mM to 12mM With the EDTA of 0.1mM to 0.2mM, the TE pH of cushioning fluid is 7.5 to 7.8.

9. method as claimed in any of claims 1 to 8, which is characterized in that the thermophilic category fungi is thermophilic Du Nation bacterium (Thermomyces dupontii) and/or thermophilic cotton wool bacterium (Thermomyces lanuginosus).

10. a kind of thermophilic identification method for belonging to (Thermomyces) fungi positive genetic transformation comprising following steps：

With thermophilic category (Thermomyces) fungi as described in the method acquisition as described in any one in claim 1 to 9 Then pcr template carries out PCR screenings.