CN113249448A - Method for accurately quantifying mycelium biomass of bag-cultivated oyster mushrooms and application of method - Google Patents
Method for accurately quantifying mycelium biomass of bag-cultivated oyster mushrooms and application of method Download PDFInfo
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Abstract
The invention discloses a method for accurately quantifying the mycelium biomass of bag-cultivated oyster mushrooms and application thereof. The method comprises the following steps: (1) amplifying and sequencing the specific phos gene of the oyster mushroom; (2) designing a phos gene fluorescent quantitative PCR primer and optimizing amplification conditions; (3) preparing oyster mushroom mycelia; (4) preparing a pleurotus ostreatus mycelium-culture material mixed sample and extracting genome DNA; (5) and (3) constructing a pleurotus ostreatus hypha DNA copy number-biomass standard curve. The method not only provides a good solution for the accurate quantification of the bag cultivation oyster mushroom hyphae, but also provides a reference for the follow-up exploration of the relationship between the oyster mushroom hypha biomass and the fruiting body yield and the disclosure of the dynamic change of the hypha biomass of the oyster mushroom in different growth and development periods.
Description
Technical Field
The invention belongs to the technical field of edible fungus hypha biomass measurement, and particularly relates to a method for accurately quantifying the hypha biomass of bag-cultured oyster mushrooms and application of the method in oyster mushroom production.
Background
The oyster mushroom is one of the main edible mushroom varieties in China, the cultivation of the oyster mushroom is influenced by external environmental factors and the nutrient composition of a culture material, and the biomass of oyster mushroom hyphae has certain difference under different cultivation conditions and different growth and development periods. At present, the description of the growth of oyster mushroom hyphae is limited to the qualitative description of the growth vigor (color, density, thickness and the like) and the growth speed of aerial hyphae, and the qualitative description of a large amount of substrate hyphae growing in a culture material cannot be accurately described. However, the degradation rate of the oyster mushroom mycelia to the culture material is mainly determined by the content of the mycelia in the substrate, and in addition, a certain correlation exists between the yield of the oyster mushroom fruiting bodies and the biomass of the mycelia. Therefore, in order to reveal the change of the hypha biomass of the oyster mushrooms under different culture conditions and at different growth and development periods, a scientific and reasonable hypha biomass quantitative evaluation system needs to be established.
At present, the DNA concentration measurement method is used for the hypha quantitative research of factory needle mushroom and chamotte cultivated mushroom, but the measurement result of the method is easily influenced by the cultivation substrate source DNA sample. Particularly, in the cultivation of oyster mushroom fermented materials, the culture materials are complex mixtures which are formed by oyster mushroom hypha, other microorganisms and cultivation substrates, and DNA extracted from the oyster mushroom fermented materials not only contains DNA from the oyster mushroom hypha, but also contains DNA from the cultivation substrates and other microorganisms. Then, when the DNA concentration quantitative method is adopted to quantify the oyster mushroom hyphae cultivated by the fermentation material, a large error is inevitably generated. Therefore, the development of a highly specific quantitative method is an effective approach to solve the above problems.
Disclosure of Invention
The invention aims to: provides a method for accurately quantifying the biomass of bag-cultivated oyster mushroom mycelia and application thereof, aiming at solving the problems of inaccurate quantification and larger error of the oyster mushroom mycelia biomass in the prior art.
The technical scheme adopted by the invention is as follows:
a method for accurately quantifying the mycelium biomass of bag-cultivated oyster mushrooms. The method comprises the following steps:
(1) pleurotus ostreatus-specific phoS gene amplification and sequencing
(2) PhoS gene fluorescent quantitative PCR primer design and amplification condition optimization
(3) Preparation of oyster mushroom hypha
(4) Preparation of oyster mushroom mycelium-culture material mixed sample and extraction of genome DNA
(5) Construction of oyster mushroom hypha DNA copy number-biomass standard curve
DNA copy numbers of hypha-culture material mixed samples with different concentrations are quantified by utilizing the specific gGSThos-F and gGSThos-R primers of the oyster mushroom through fluorescent quantitative PCR, and a standard curve between the hypha DNA copy number and biomass is established.
Further, the step (1) specifically comprises the following steps:
firstly, designing a primer to amplify a phoS gene segment specific to the oyster mushroom;
constructing pMD19T-phoS recombinant plasmid;
sequencing pMD19T-PhoS recombinant plasmid, wherein the sequence of PhoS gene is shown in SEQ ID NO. 1.
Further, the step (2) specifically comprises the following steps:
designing an upstream primer gGSThos-F positioned in an intron according to the sequence of the PhoS gene and the design principle of a fluorescent quantitative PCR primer: 5 '… GTCTCGCCCCTTTATCTGAATG … 3', downstream primer gRTphoS-R located in exon: 5 '… GGACTTGAACGCTGCGATATTTG … 3';
and secondly, screening and optimizing the annealing temperature of the gPRoS-F/gPRoS-R primer by using temperature gradient PCR. Furthermore, in the step II, the annealing temperature is 53-58 ℃, and preferably 58 ℃.
Further, the step (3) specifically includes the steps of: transferring the activated liquid strain of the oyster mushroom to a liquid culture medium, carrying out shake culture in the dark, filtering and collecting mycelia, and washing with sterile water. Further, the strain is a new strain of Pleurotus ostreatus 831.
Further, the step (4) specifically includes the steps of: evenly mixing oyster mushroom mycelia with different wet weights and a culture material with a constant weight, preparing a mycelium-culture material mixed sample, quickly grinding the mycelium-culture material mixed sample into powder under the condition of liquid nitrogen, and extracting the genomic DNA of the mixed sample by adopting a CTAB method.
Further, the step (5) specifically includes the steps of:
construction of PhoS gene absolute fluorescent quantitative PCR standard curve
Taking the pMD19T-phoS plasmid constructed in the step (1) as a standard plasmid, determining the copy number of the phoS gene under each dilution by real-time fluorescent quantitative PCR, and establishing a standard curve between log10 (copy number) and Ct value;
② construction of Pleurotus ostreatus hypha DNA copy number-biomass standard curve
And (3) taking the genomic DNA of each sample extracted in the step (4) as a template, determining the Ct value of the phoS gene in each sample by using a gGSThos-F/gGSThos-R primer, substituting the Ct value into a log10 (copy number) -Ct value standard curve to calculate the DNA copy number of each sample, and establishing a standard curve between the DNA copy number of the pleurotus ostreatus hyphae and the biomass.
Further, the step (i) specifically includes the steps of: a. extracting pMD19T-phoS plasmid, and determining the concentration of the plasmid; b. the extracted pMD19T-phoS plasmid was diluted in 10-fold gradients, and the DNA copy number of each dilution sample was calculated and further the log10 (copy number) value was calculated; c. taking the diluted pMD19T-phoS plasmid as a template, performing fluorescent quantitative PCR amplification by using a gPRoS-F/gPRoS-R primer, determining the Ct value of each diluted sample, and repeating each sample for three times; d. a standard curve between log10 (copy number) and Ct values was established.
Specifically, wherein: the culture medium comprises 70% of cottonseed hull and 30% of wheat bran, and phosphorus elements of 0%, 0.2%, 0.4%, 0.6%, 1.0% and 1.2% (W/W) are added respectively, and the water content is about 60%.
Wherein: the manufacturing steps of the material bag are as follows: accurately weighing the cottonseed hulls and the wheat bran according to the formula proportion, and uniformly reprinting; accurately weighing disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dodecahydrate according to a formula, adding the disodium hydrogen phosphate dihydrate and the sodium dihydrogen phosphate dodecahydrate into weighed warm water, and stirring for dissolving; adding the dissolved phosphate solution into a mixed sample of the cottonseed hulls and the wheat bran, and stirring uniformly; filling into polypropylene angular bags of 22cm × 36cm × 0.004cm, and sealing with collars; sterilizing at 100 deg.C under normal pressure for 16 h; after the material bag is cooled, inoculating oyster mushroom solid strains at one end under an aseptic condition, and sealing the strains; the bags are moved to a 25 ℃ culture room and cultured until the mycelium of a certain treatment group grows to half a bag.
Wherein: the qualitative analysis of the hypha growth vigor is as follows: opening a fungus bag at a position 1cm away from the tip of the hypha, and photographing to record the hypha growth of the cross section;
wherein: the hypha genome DNA extraction steps are as follows: kneading and crushing the culture material with the mycelium ends, and randomly weighing 5 g; quickly freezing with liquid nitrogen, and grinding into powder; weighing 0.2g of powder, and extracting genome DNA by using a CTAB method;
wherein: the quantitative steps of the pleurotus ostreatus hypha biomass in the culture material are as follows: the Ct value of the phoS gene in each sample is determined by utilizing a gGSThos-F/gGSThos-R primer, the Ct value is substituted into a log10 (copy number) -Ct value standard curve, a log10 (copy number) is obtained by calculation, and the DNA copy number is further calculated; substituting the DNA copy number into a standard curve of the Pleurotus ostreatus hypha DNA copy number-biomass, and calculating to obtain the dry weight of the Pleurotus ostreatus hypha.
In addition, the invention also provides application of the method for accurately quantifying the mycelium biomass of the bag-cultured oyster mushrooms in oyster mushroom production.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the phoS gene of the oyster mushroom strain is used as a target gene, and an absolute fluorescent quantitative PCR standard curve of the phoS gene is established through fluorescent quantitative PCR. A mixed sample containing different biomass mycelia is prepared by uniformly mixing oyster mushroom mycelia with different weights and a culture material with a constant weight so as to simulate an oyster mushroom bag cultivation experiment. Then, genomic DNA of each sample was extracted, and the copy number of the phoS gene in each genomic sample was determined by fluorescent quantitative PCR. And finally, converting and establishing a standard curve between hyphal biomass and DNA copy number. The research result of the invention not only provides a good method for the accurate quantification of oyster mushroom hypha cultivated by clinker or fermented material, but also provides a reference for the follow-up research of the relationship between the oyster mushroom hypha biomass and the fruiting body yield and the disclosure of the dynamic change of the hypha biomass of oyster mushroom in different growth and development periods.
Drawings
FIG. 1 PhoS Gene gDNA fragment amplification electropherogram (A) and pMD19T-PhoS recombinant plasmid electropherogram (B), (A) M, Tans2K Plus II DNA marker; 1 and 2, phoS gene gDNA fragment, (B) M, Tans2K Plus II DNA marker; 1, pMD19-T unloaded plasmid; 2 and 3, pMD19T-phoS recombinant plasmids;
FIG. 2 design principle of fluorescent quantitative PCR primers for PhoS gene;
FIG. 3 gPROS-F/gPROS-R primer annealing temperature Screen, M, Tans2K Plus II DNA marker; 1 to 3, the annealing temperature is 53 ℃, 56 ℃ and 58 ℃ and the amplification result of the gDNA fragment is obtained; 4 to 6, and the amplification result of the primer dimer is obtained when the annealing temperature is 53 ℃, 56 ℃ and 58 ℃;
FIG. 4 PhoS gene absolute fluorescent quantitative PCR standard curve;
FIG. 5 is a broad range standard curve between Pleurotus ostreatus hypha DNA copy number and biomass;
FIG. 6 is a small scale standard curve between Pleurotus ostreatus hypha DNA copy number and biomass;
FIG. 7 is a test for the accuracy of the Pleurotus ostreatus hypha DNA copy number-biomass standard curve;
FIG. 8 application of the Pleurotus ostreatus hypha DNA copy number-biomass standard curve.
Detailed Description
The present invention will be further described with reference to the following embodiments. The present invention is not limited to the following examples.
The phoS gene of the new strain 831 of the oyster mushroom is used as a target gene, the oyster mushroom hyphae are used as a test material, an absolute fluorescent quantitative PCR standard curve of the phoS gene is established by a fluorescent quantitative PCR method, and a linear relation between the DNA copy number and the biomass of the oyster mushroom hyphae is further established. The test result shows that the absolute fluorescent quantitative PCR standard curve of the oyster mushroom phoS gene is y-2.8917 x +34.231, the standard curve (large range) between the oyster mushroom DNA copy number and hypha biomass is y-0.2973 x-0.33347, and R is20.9987, the standard curve (small range) between the oyster mushroom DNA copy number-hyphal biomass is y ═ y0.1719x-0.0.039,R20.993. The pleurotus ostreatus mycelium biomass and the DNA copy number have good linear relation, can be used for accurately quantifying the pleurotus ostreatus mycelium biomass in bag cultivation, and is particularly suitable for measuring the pleurotus ostreatus mycelium biomass in fermented material cultivation.
Examples
(I) test materials
Test strains and plasmids
The new 831 strain of oyster mushroom is preserved in the laboratory, and the pMD19T-phos plasmid is constructed in the invention.
Culture medium
(1) GYE liquid medium: 2% of glucose, 0.5% of yeast powder and 0.1% of KH2PO4、0.05％MgSO4·7H2O、0.01‰VB1And 1L of distilled water. Sterilizing with high pressure steam at 115 deg.C for 30 min.
(2) The formula of the pure cotton seed hull culture material comprises the following components: 100% of cottonseed hull, and the water content is 60%.
(3) The formula of the phosphorus-containing culture material comprises: respectively adding phosphorus elements with different concentrations into 70% of cottonseed hulls and 30% of wheat bran to prepare culture mediums with the phosphorus element contents of 0%, 0.2%, 0.4%, 0.6%, 1.0% and 1.2%, and the water content is 60%. Sterilizing at 100 deg.C under normal pressure for 16 h.
(II) test method
1. Amplification and sequencing of the Pleurotus Ostreatus-specific PhoS Gene
(1) Primers for amplifying phoS gene of the new 831 strain of oyster mushroom are designed according to the genome sequence of P.ostreatus PC15, and the primer sequences are as follows:
phoS-F:5'...GGTGATAGTATTGATCGGCGTTATTG...3'
phoS-R:5'...CATGATCGAGAACTTCATGGAAC...3'
(2) the genome DNA of the new 831 strain of the oyster mushroom is used as a template for amplification, and the reaction system is as follows: ddH2O34. mu.L, 5 XPS Buffer 10. mu.L, dNTPs 4. mu.L, phoS-F0.5. mu.L, phoS-R0.5. mu.L, genomic DNA 0.5. mu.L, Primerstar DNA polymerase 0.5. mu.L.
The reaction conditions are as follows: 3min at 98 ℃; 30 cycles of 98 ℃ for 10s, 58 ℃ for 15s, and 72 ℃ for 1min for 10 s; 5min at 72 ℃.
(3) Performing gel recovery on the PCR product according to the specification of the Shanghai raw gel recovery kit;
(4) performing enzyme-linked reaction according to the kit instructions of pMD19-T vector (TAKARA);
(5) carrying out Escherichia coli transformation and pMD19T-phoS recombinant plasmid screening according to the conventional molecular cloning steps;
(6) the pMD19T-phoS recombinant plasmid was sequenced from the Huahuada gene (the sequence of the phoS gene is shown in SEQ ID NO. 1).
Design of 2 phoS gene specific fluorescent quantitative PCR amplification primer and annealing temperature screening
2.1 amplification primer design
According to the primer design principle shown in FIG. 2, the real-time fluorescent quantitative PCR amplification primer of PhoS gene specificity is designed according to the sequencing result of pMD19T-PhoS recombinant plasmid, wherein, the upstream primer gGSTPhoS-F is positioned in an intron, and the downstream primer gGSTPhoS-R is positioned in an exon, thereby eliminating the influence of RNA on template amplification. The primer sequences are as follows:
gRTphoS-F：5’…GTCTCGCCCCTTTATCTGAATG…3’
gRTphoS-R:5’…GGACTTGAACGCTGCGATATTTG…3’
2.2 amplification annealing temperature screening
The reaction system is as follows: ddH2O20. mu.L, 10 XBuffer 2.5. mu.L, dNTP 2. mu.L, gPRoS-F0.4. mu.L, gPRoS-R0.4. mu.L, gDNA 0.25. mu.L. Negative control group did not add gDNA.
Three annealing temperatures of 53 ℃, 56 ℃ and 58 ℃ are set. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 53 ℃ and 56 ℃ and 58 ℃ for 30 seconds, extension at 72 ℃ for 20 seconds, and 35 cycles; the temperature was maintained at 72 ℃ for 5 minutes. After the PCR was completed, 5. mu.L of the amplified product was detected by 1.2% agarose gel electrophoresis (FIG. 3).
3. Preparation of oyster mushroom hypha
(1) Transferring the activated oyster mushroom liquid strain into GYE liquid culture medium according to the inoculation amount of 5%, and culturing at 25 ℃ and 180rpm in dark for about 5 days with shaking.
(2) The mycelium was collected by vacuum filtration and washed once with sterile water.
Preparation of 4 oyster mushroom mycelium-culture material mixed sample and extraction of genome DNA
4.1 preparation of Pleurotus Ostreatus mycelium-culture Material Mixed sample
(1) In order to simulate the living environment under the cultivation condition of oyster mushroom mycelia, oyster mushroom mycelia (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.3, 0.6 and 1.5g) with different wet weights are uniformly mixed with a pure cotton seed shell culture material with a constant weight (4g) to prepare a mycelia-culture material mixed sample, and the treatment is repeated three times each time. (bag cultivation oyster mushroom hypha biomass is particularly low and cannot exceed 1.5 g.)
(2) And adding the hypha-culture material mixed sample into a mortar precooled by liquid nitrogen, pouring liquid nitrogen with a proper volume, and quickly grinding the sample into powder.
(3) Measurement of the dry weight of the hyphae: accurately weighing 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.3, 0.6 and 1.5g of oyster mushroom mycelia, putting the oyster mushroom mycelia into a 65 ℃ oven, drying to constant weight, and weighing the dry weight of the mycelia.
(4) Determination of dry weight of pure culture material: accurately weighing 4g of pure cotton seed hull culture material, putting the pure cotton seed hull culture material into a 65 ℃ oven, drying the pure cotton seed hull culture material to constant weight, and weighing the dry weight of the pure culture material.
4.2 extraction of genomic DNA from Pleurotus Ostreatus hypha
The invention adopts a CTAB method to extract DNA of a hypha-culture material mixed sample.
(1) Preparation of the solution
1)2 × CTAB buffer (500 mL): 2% CTAB, 100mM Tris-HCl, 20mM EDTA, 1.4M NaCl, 1% pH8.0 polyvinylpyrrolidone (PVP).
2) Soil DNA extraction buffer (500 mL): 100mM NaCl, 50mM EDTA, 0.25M Tris-HCl, 5% SDS, sterilized and stored at room temperature.
3) TE buffer: 10mM Tris-HCl, 1mM EDTA (pH8.0), prepared, sterilized at 121 ℃ for 20min in a vertical autoclave, and stored at room temperature.
4) TE buffer (stabilizing RNase a 1): mu.L of RNase A1(50mg/mL) was added to 1mL TE buffer to give a final concentration of RNase A1 of 1 mg/mL. And (4) preserving in a refrigerator at the temperature of minus 20 ℃.
5)3M sodium acetate solution (pH5.2) (100 mL): weighing 24.61g of anhydrous sodium acetate, adding 80mL of ultrapure water, adjusting the pH to 5.2 with glacial acetic acid, diluting to 100mL, sterilizing at 121 ℃ for 20min, and storing at room temperature.
6) 70% ethanol (200 mL): 140mL of absolute ethyl alcohol (analytically pure) is weighed into a reagent bottle, 60mL of sterilized ultrapure water is added, and the mixture is stored in a refrigerator at 4 ℃ for later use.
(2) Extraction of oyster mushroom hypha genome DNA
1) Accurately weigh 0.2g of the sample pulverized with liquid nitrogen into a 2mL centrifuge tube precooled with liquid nitrogen.
2) Add 400. mu.L of Soil DNA extraction buffer, reverse vigorously and mix by vortexing.
3) Adding 400mL of 2 xCTAB buffer solution, violently reversing and mixing by vortex oscillation; standing at 55 deg.C for 5min to fully lyse cells.
4) Adding 500 μ L of phenol, chloroform and isoamyl alcohol (25:24:1), violently reversing and mixing by vortex shaking; centrifuge at 12,000rpm, 4 ℃ for 10 min.
5) Transferring about 650 mu L of supernatant into a 1.5mL centrifuge tube by using a pipette; adding 455 mu L of isopropanol, reversing and uniformly mixing to precipitate DNA; centrifuge at 12,000rpm at 4 ℃ for 10min and discard the supernatant.
6) Adding 1mL of precooled 70% ethanol, and performing vortex oscillation; centrifuging at 4 deg.C and 12,000rpm for 10min, and removing supernatant; and (5) placing in a fume hood for 3-5 min to remove the alcohol.
7) Adding 50 μ L of preheated TE buffer (stabilizing RNase A1) to dissolve the DNA precipitate, incubating at 65 deg.C for 10min, and inactivating the DNase; incubate at 37 ℃ for 30min to remove RNA from the DNA sample.
8) Adding 5 μ L of 3M sodium acetate buffer (pH5.2) and 125 μ L of absolute ethanol, and standing in a refrigerator at-80 deg.C for 30 min; centrifuging at 4 deg.C and 12,000rpm for 10min, and removing supernatant;
9) repeating step 6);
10) add 50. mu.L of a 65 ℃ preheated TE buffer and flick to dissolve the pellet in buffer and store in the freezer at-80 ℃.
5 establishment of standard curve between DNA copy number and biomass of oyster mushroom hypha
Construction of 5.1 PhoS Gene Absolute fluorescent quantitative PCR Standard Curve
(1) Extracting pMD19T-phoS plasmid by using a Shanghai biological medium particle small quantity extraction kit, and determining the concentration of the plasmid;
(2) the extracted pMD19T-phoS plasmid was diluted in a 10-fold gradient (10)1、102、103、104、105、106、107、108、109)；
(3) Fluorescent quantitative PCR amplification was performed using the gPRoS-F and gPRTPoS-R primers, using the diluted pMD19T-phoS plasmid as a template, and each sample was replicated three times.
The reaction system is as follows: 2 XSSYB Green 10. mu.L, different dilutions of pMD19T-phoS plasmid 0.5. mu.L, gPRoS-F0.4. mu.L, gPRoS-R0.4. mu.L, ddH2O 9μL。
The reaction conditions are as follows: 2min at 95 ℃; 40 cycles of 95 ℃ for 20s, 56 ℃ for 20s, 72 ℃ for 20 s; 95 ℃ for 5s, 4.4 ℃/s; 1min at 65 ℃ and 2.2 ℃/s; 65 ℃ to 95 ℃ continuos, 0.1 ℃/s; cool at 40 ℃ cool, 2.2 ℃/s.
(4) PhoS Gene Standard Curve preparation
The copy number of the pMD19T-phoS plasmid was calculated for each dilution, and a standard curve of the phoS gene was plotted with log10 (copy number) as abscissa and Ct value as ordinate (FIG. 4).
5.2 construction of Pleurotus Ostreatus hypha DNA copy number-Biomass Standard Curve
(1) Fluorescent quantitative PCR amplification was performed using 1. mu.L of sample DNA as template and gGSThos-F/gGSThos-R primers, and each sample was repeated three times.
(2) Substituting the Ct value corresponding to each sample into the PhoS gene absolute fluorescence quantitative standard curve to calculate the log10 (copy number) value of the PhoS gene of each sample; the number of copies of the phoS gene in each sample was further calculated. In order to eliminate the difference of water content of hyphae of different samples, the invention characterizes hyphae biomass by hyphae dry weight. A DNA copy number-Pleurotus ostreatus hypha biomass standard curve was plotted with the hypha dry weight as abscissa and the DNA copy number as ordinate (FIGS. 5 and 6).
6 accuracy test of standard curve between DNA copy number and biomass of oyster mushroom hyphae
Accurately weighing 0g (T1), 0.4g (T2), 1g (T3) and 2g (T4) of oyster mushroom mycelia and 4g of culture materials, uniformly mixing to prepare oyster mushroom bran, weighing 0.2g of uniformly mixed culture materials, and extracting genome DNA. Fluorescent quantitative PCR amplification was performed as described above. The Ct values of the phoS genes of the samples are sequentially substituted into a phoS gene standard curve to calculate the copy number of the phoS genes, the copy number of the phoS genes is further substituted into a DNA copy number-hypha biomass standard curve to calculate the dry weight of the oyster mushroom hyphae in the oyster mushroom bran, and the dry weight of the oyster mushroom hyphae is compared with the actual dry weight of the oyster mushroom hyphae (figure 7).
The positive correlation exists between the oyster mushroom hypha dry weight calculated by utilizing the phoS gene absolute fluorescence quantitative standard curve and the oyster mushroom hypha DNA copy number-biomass standard curve established by the invention and the actual oyster mushroom hypha dry weight, so that the method for accurately quantifying the oyster mushroom hypha biomass by the bag cultivation method established by the invention has high accuracy and can be widely applied to oyster mushroom production. Compared with the traditional method for measuring the DNA concentration and the protein concentration of the hyphae and the like, the method for quantifying the hyphae of the oyster mushroom can eliminate the interference of the DNA of a culture material and the DNA of other microorganisms, and can accurately quantify the biomass of the hyphae of the oyster mushroom.
Application of standard curve between 7 oyster mushroom hypha DNA copy number and biomass
(1) Material bag manufacturing
1) Accurately weighing the cottonseed hulls and the wheat bran according to the formula proportion, and stirring uniformly;
2) accurately weighing disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dodecahydrate according to a formula, adding the disodium hydrogen phosphate dihydrate and the sodium dihydrogen phosphate dodecahydrate into weighed warm water, and stirring for dissolving;
3) adding the dissolved phosphate solution into a mixed sample of the cottonseed hulls and the wheat bran, and stirring uniformly;
4) filling the culture materials into polypropylene angular bags of 22cm multiplied by 36cm multiplied by 0.004cm, and sealing the lantern rings;
5) sterilizing at 100 deg.C under normal pressure for 16 h;
6) after the material bag is cooled, inoculating oyster mushroom solid strains at one end under an aseptic condition, and sealing the strains;
7) the bags are moved to a 25 ℃ culture room and cultured until the mycelium of a certain treatment group grows to half a bag.
(2) Qualitative analysis of hypha growth
And (5) opening the fungus bag at a position 1cm away from the hypha tip, and photographing to record the hypha growth of the cross section.
(3) Hyphal genomic DNA extraction
1) Kneading and crushing the culture material with the mycelium ends, and randomly weighing 5 g;
2) quickly freezing with liquid nitrogen, and grinding into powder;
3) accurately weighing 0.2g of powder into a centrifugal tube, and extracting the genome DNA by using a CTAB method.
(4) Quantitative determination of oyster mushroom hypha biomass in culture material
1) The Ct value of the phoS gene in each sample is determined by using gGSThos-F/gGSThos-R primers;
2) the Ct value is substituted into a phoS gene absolute fluorescence quantitative standard curve, and log10 (copy number) is obtained through calculation;
3) further calculating the DNA copy number;
4) substituting the DNA copy number into a standard curve of the Pleurotus ostreatus hypha DNA copy number-biomass, and calculating to obtain dry weight of the Pleurotus ostreatus hypha;
5) calculating the dry weight ratio of the culture material: accurately weighing 100g of the culture material, putting the culture material into a 65 ℃ oven, drying to constant weight, weighing the dry weight of the culture material, and calculating the dry weight ratio;
6) the mycelium biomass of the pleurotus ostreatus of each treatment group is normalized by the dry weight of the compost to eliminate errors caused by different water contents of the compost, and finally the dry weight (mg/g dry material) of the mycelium is obtained (figure 8).
The phoS gene absolute fluorescence quantitative standard curve and the oyster mushroom hypha DNA copy number-biomass standard curve which are established by the invention are used for calculating to obtain the oyster mushroom hypha dry weight which is in positive correlation with the oyster mushroom hypha growth vigor which is qualitatively observed, so that the method for accurately quantifying the oyster mushroom hypha biomass by bag cultivation, which is established by the invention, has high accuracy and can be widely applied to oyster mushroom production. Compared with the traditional method for measuring the DNA concentration and the protein concentration of the hyphae and the like, the method for quantifying the hyphae of the oyster mushroom can eliminate the interference of the DNA of a culture material and the DNA of other microorganisms, and can accurately quantify the biomass of the hyphae of the oyster mushroom.
In addition, the invention respectively constructs a large-range standard curve (figure 5) and a small-range standard curve (figure 6) between the DNA copy number and the biomass of the oyster mushroom hypha, and can select a proper standard curve to calculate the biomass of the oyster mushroom hypha according to the actually measured DNA copy number.
Sequence listing
<110> Henan university of agriculture
<120> method for accurately quantifying mycelium biomass of bag-cultured oyster mushrooms and application thereof
<141> 2021-03-05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1003
<212> DNA
<213> oyster Mushroom (Pleurotus ostreatus)
<400> 1
ggtgatagta ttgatcggcg ttattggcgg tagcggcctg tatcacttgg ataacttgtc 60
gatcgtgtaa gtgagcgatt tatgttgctg cccgcggtta ctgattttga atgataggaa 120
ggaagtcaat ccagagactg tacggtctcg cccctttatc tgaatggctg gctggctgac 180
cctcgattag ccctggggct tcccaagctc tccgatcatc atctgtgccc tcccttctgg 240
caccaaagtc gctttcctcg cgcgacatgg gcagggacac atccacgcac cgtctgccgt 300
gccagctcgc gcaaatatcg cagcgttcaa gtccctcggc gtgcgcgcga tcttggcctt 360
ctcagcggtg ggctccttgc gcgaggaaat atccccagga tcctttgtcc tcccatcgca 420
aatcatcgat cgcaccaagg gcgtgcgccc ggcgagcttc ttcgagggga ccagcatcgt 480
ggcccacgct gcgtttggcg atccgttcag caacaaattg gtgcattggc tggagagccg 540
cgtgcagaag gcgttggatg cggagggacg cggtgtgcag ctattcactg ggaagacgat 600
cgtctgcatg gagggacctc agttctctac gagagcagag agcattatgt accgccagtg 660
gggaggggac ttgattaaca tgagcgtcct ccctgaagca aagctggctc gcgaggccga 720
gttgaggtac gtctttcttc tcgtatggtc tccttcgggt gctcagcagt gtcgtagcta 780
cgcgcttgta gccaccgcta cggactacga ttcgtggcgc ccccaatctg aggcggtcac 840
agcgatggag gtgttcaaaa cattgcaggc aaacgctgac acatcgcgcc atgtctgcgc 900
tacgattctc gacgagctgc atgaagccgt cgacaataac gctgcctcgg aagatgccga 960
tatcttatgt gaggaagtgg gttccatgaa gttctcgatc atg 1003
Claims (10)
1. A method for accurately quantifying the mycelium biomass of bag-cultivated oyster mushrooms is characterized by comprising the following steps:
(1) pleurotus ostreatus-specific phoS gene amplification and sequencing
(2) PhoS gene fluorescent quantitative PCR primer design and amplification condition optimization
(3) Preparation of oyster mushroom hypha
(4) Preparation of oyster mushroom mycelium-culture material mixed sample and extraction of genome DNA
(5) Construction of oyster mushroom hypha DNA copy number-biomass standard curve
DNA copy numbers of hypha-culture material mixed samples with different concentrations are quantified by utilizing the specific gGSThos-F and gGSThos-R primers of the oyster mushroom through fluorescent quantitative PCR, and a standard curve between the hypha DNA copy number and biomass is established.
2. The method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushroom according to claim 1, wherein the step (1) comprises the following steps:
firstly, designing a primer to amplify a phoS gene segment specific to the oyster mushroom;
constructing pMD19T-phoS recombinant plasmid;
sequencing pMD19T-PhoS recombinant plasmid, wherein the sequence of PhoS gene is shown in SEQ ID NO. 1.
3. The method for accurately quantifying the hypha biomass of the bag-cultivated pleurotus ostreatus according to claim 1, wherein in the step (2), the fluorescent quantitative PCR primer of the gene gRT _ PCR is designed in such a way that an upstream primer gRT _ Pho-F is positioned in an intron, and a downstream primer gRT _ Pho-R is positioned in an exon.
4. The method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushrooms according to claim 3, wherein in the step (2), the sequence of upstream primer gGSThos-F is 5 '… GTCTCGCCCCTTTATCTGAATG … 3', and the sequence of downstream primer gGSThos-R is 5 '… GGACTTGAACGCTGCGATATTTG … 3'.
5. The method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushrooms according to claim 4, wherein in the step (2), the annealing temperature of gPRoS-F/gPRoS-R primers is 53-58 ℃.
6. The method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushrooms according to claim 5, wherein in the step (2), the annealing temperature of gPRoS-F/gPRoS-R primers is 58 ℃.
7. The method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushroom according to claim 1, wherein the step (4) comprises the following steps: evenly mixing oyster mushroom mycelia with different wet weights and a culture material with a constant weight, preparing a mycelium-culture material mixed sample, quickly grinding the mycelium-culture material mixed sample into powder under the condition of liquid nitrogen, and extracting the genomic DNA of the mixed sample by using a CTAB method.
8. The method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushroom according to claim 2, wherein the step (5) comprises the following steps:
construction of PhoS gene absolute fluorescent quantitative PCR standard curve
Taking the pMD19T-phoS plasmid constructed in the step (1) as a standard plasmid, determining the Ct value of the phoS gene under each dilution by real-time fluorescent quantitative PCR, and establishing a standard curve between the copy number and the Ct value;
② construction of Pleurotus ostreatus hypha DNA copy number-biomass standard curve
And (4) taking the genomic DNA of each sample extracted in the step (4) as a template, determining the Ct value of the phoS gene in each sample by using the gPROS-F/gPROS-R primers, substituting the Ct value into a copy number-Ct value standard curve to calculate the DNA copy number of each sample, and establishing a standard curve between the DNA copy number of the oyster mushroom hyphae and the biomass.
9. The method for accurately quantifying the biomass of bag-cultured oyster mushroom mycelia according to claim 8, wherein the step (r) specifically comprises the steps of: a. extracting pMD19T-phoS plasmid, and determining the concentration of the plasmid; b. carrying out 10-fold gradient dilution on the extracted pMD19T-phoS plasmid, calculating the DNA copy number of each dilution sample, and further calculating the copy number; c. taking the diluted pMD19T-phoS plasmid as a template, performing fluorescent quantitative PCR amplification by using a gPRoS-F/gPRoS-R primer, determining the Ct value of each diluted sample, and repeating each sample for three times; d. a standard curve between copy number and Ct value was established.
10. Use of the method for accurately quantifying the biomass of mycelia of bag-cultured oyster mushrooms according to any one of claims 1 to 9 in the production of oyster mushrooms.
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| RAÚL CASTANERA ET AL.: ""Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR"", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》, vol. 81, no. 12, pages 4120 - 4129 * |
| 邢楠: ""Real-time PCR用于土壤中稻纹枯病原菌的定量监测"", 《硕士电子期刊》, no. 4, pages 1 - 54 * |
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