CN113249448B - Method for accurately quantifying bag-cultivated oyster mushroom mycelium biomass and application thereof - Google Patents
Method for accurately quantifying bag-cultivated oyster mushroom mycelium biomass and application thereof Download PDFInfo
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Abstract
The invention discloses a method for accurately quantifying bag-cultivated oyster mushroom mycelium biomass and application thereof. The method comprises the following steps: (1) oyster mushroom-specific phos gene amplification and sequencing; (2) The fluorescent quantitative PCR primer design and the amplification condition optimization of the phos gene; (3) oyster mushroom hypha preparation; (4) Preparing an oyster mushroom mycelium-culture material mixed sample and extracting genome DNA; (5) And constructing an oyster mushroom hypha DNA copy number-biomass standard curve. The method not only provides a good solution for the accurate quantification of bag cultivated oyster mushroom mycelia, but also provides a reference for the follow-up exploration of the relation between oyster mushroom mycelia biomass and fruiting body yield and the disclosure of dynamic changes of the oyster mushroom mycelia biomass in different growing and developing periods.
Description
Technical Field
The invention belongs to the technical field of edible fungus mycelium biomass measurement, and particularly relates to a method for accurately quantifying bag-cultivated oyster mushroom mycelium biomass and application of the method in oyster mushroom production.
Background
Oyster mushroom is one of main cultivated edible fungus varieties in China, the cultivation of the oyster mushroom is influenced by external environmental factors and nutrient composition of culture materials, and the biomass of oyster mushroom hyphae has certain difference under different cultivation conditions and at different growth and development periods. At present, the description of the growth of oyster mushroom mycelia is limited to qualitative description of the growth vigor (hypha color, density, thickness and the like) and the growth speed of aerial hypha, and the qualitative description of a large number of basal hypha growing inside a culture material still cannot be accurate. However, the degradation rate of oyster mushroom mycelia on the culture medium mainly depends on the content of mycelia in the substrate, and in addition, there is a certain correlation between the yield of oyster mushroom fruiting bodies and the biomass of mycelia thereof. Therefore, in order to reveal the variation of the mycelium biomass of oyster mushrooms under different culture conditions and at different growth and development periods, a scientific and reasonable mycelium biomass quantitative evaluation system needs to be established.
At present, a DNA concentration measurement method is used for quantitative study of mycelia of industrial flammulina velutipes and chamotte cultivated mushrooms, but the measurement result of the method is easily influenced by a DNA sample derived from a cultivation substrate. Especially in oyster mushroom ferment cultivation, the culture material is a complex mixture composed of oyster mushroom hypha, other microorganisms and culture medium, and DNA extracted from the oyster mushroom ferment contains both oyster mushroom hypha-derived DNA and culture medium and other microorganism-derived DNA. Then a DNA concentration quantitative method is adopted to quantitatively determine the oyster mushroom hypha cultivated by the fermentation material, so that a larger error is necessarily generated. Therefore, developing a highly specific quantitative method is an effective approach to solve the above-mentioned problems.
Disclosure of Invention
The invention aims at: provides a method for precisely quantifying the biomass of oyster mushroom mycelia in bag cultivation and application thereof, and aims to solve the problems of inaccurate biomass quantification of oyster mushroom mycelia and larger error in the prior art.
The technical scheme adopted by the invention is as follows:
a method for accurately quantifying the biomass of bag-cultivated oyster mushroom hypha. The method comprises the following steps:
(1) Oyster mushroom specific phoS gene amplification and sequencing
(2) PhoS gene fluorescent quantitative PCR primer design and amplification condition optimization
(3) Oyster mushroom mycelium preparation
(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 mycelium-culture mixture samples of different concentrations were quantified by fluorescent quantitative PCR using Ping Gu specific gRTphos-F and gRTphos-R primers, and a standard curve between mycelium DNA copy numbers and biomass was established.
Further, the step (1) specifically includes the following steps:
(1) designing a primer to amplify oyster mushroom specific phoS gene segment;
(2) constructing a pMD19T-phoS recombinant plasmid;
(3) the pMD19T-phoS recombinant plasmid was sequenced and the sequence of the phoS gene was shown in SEQ ID NO. 1.
Further, the step (2) specifically includes the following steps:
(1) the upstream primer gRTphos-F in the intron was designed according to the sequence of the phoS gene and the design principle of fluorescent quantitative PCR primers: 5'… GTCTCGCCCCTTTATCTGAATG …', downstream primer gRTphos-R in exon: 5'… GGACTTGAACGCTGCGATATTTG …';
(2) the annealing temperature of the gRTphos-F/gRTphos-R primer was optimized by temperature gradient PCR. Further, in the step (2), the annealing temperature is 53 to 58 ℃, preferably 58 ℃.
Further, the step (3) specifically includes the following steps: transferring activated oyster mushroom liquid strain to liquid culture medium, shake culturing in dark, filtering to collect mycelium, and washing with sterile water. Further, the strain is a novel 831 strain of oyster mushroom.
Further, the step (4) specifically includes the following steps: and uniformly mixing oyster mushroom mycelia with different wet weights and culture materials with constant weights, preparing a mycelia-culture material mixed sample, rapidly grinding the mixture into powder under the condition of liquid nitrogen, and extracting genome DNA of the mixed sample by adopting a CTAB method.
Further, the step (5) specifically includes the following steps:
(1) construction of absolute fluorescence quantitative PCR standard curve of phoS gene
Taking the pMD19T-phoS plasmid constructed in the step (1) as a standard plasmid, determining the copy number of the phoS gene at each dilution by real-time fluorescence quantitative PCR, and establishing a standard curve between log10 (copy number) and Ct value;
(2) construction of oyster mushroom hypha DNA copy number-biomass standard curve
And (3) taking the genomic DNA of each sample extracted in the step (4) as a template, measuring the Ct value of the phoS gene in each sample by using gRTphoS-F/gRTphoS-R primers, taking the Ct value into a log10 (copy number) -Ct value standard curve, calculating the DNA copy number of each sample, and establishing a standard curve between the oyster mushroom hypha DNA copy number and biomass.
Further, the step (1) specifically includes the following steps: a. extracting the pMD19T-phoS plasmid and determining the plasmid concentration; b. performing 10-fold gradient dilution on the extracted pMD19T-phoS plasmid, calculating the DNA copy number of each dilution sample, and further calculating a log10 (copy number) value; c. performing fluorescent quantitative PCR amplification by using the diluted pMD19T-phoS plasmid as a template and using gRTphos-F/gRTphos-R primers, and determining Ct values of samples of each dilution, wherein each sample is repeated three times; d. a standard curve between log10 (copy number) and Ct value was established.
Specifically, wherein: the culture medium comprises 70% cotton seed hull and 30% wheat bran, 0%, 0.2%, 0.4%, 0.6%, 1.0%, 1.2% (W/W) phosphorus element, and water content about 60%.
Wherein: the material bag manufacturing steps are as follows: accurately weighing cotton seed hulls and wheat bran according to the formula proportion, and uniformly turning; 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 dissolution; adding the dissolved phosphate solution into the mixed sample of cotton seed hulls and wheat bran, and stirring uniformly; placing into 22cm×36cm×0.004cm polypropylene corner bag, and sealing with collar; sterilizing at 100 ℃ under normal pressure for 16 hours; after the material bag is cooled, under aseptic condition, one end is connected with oyster mushroom solid strain, and the strain is covered; the fungus bags are carried to a culture room at 25 ℃ and cultured until hyphae of a certain treatment group grow to half bags.
Wherein: the qualitative analysis of hypha growth vigor is as follows: the fungus bag is opened at a position 1cm away from the tip of the mycelium, and the cross section mycelium growth vigor is recorded by photographing;
wherein: the mycelium genome DNA extraction steps are as follows: crushing the culture material with mycelium ends, and randomly weighing 5g; quick freezing with liquid nitrogen, and grinding into powder; weighing 0.2g of powder, and extracting genome DNA by using a CTAB method;
wherein: the oyster mushroom mycelium biomass quantification step in the culture material comprises the following steps: determining the Ct value of the phoS gene in each sample by using the gRTphos-F/gRTphos-R primer, substituting the Ct value into a log10 (copy number) -Ct value standard curve, calculating to obtain log10 (copy number), and further calculating the DNA copy number; substituting the DNA copy number into a oyster mushroom mycelium DNA copy number-biomass standard curve, and calculating to obtain the oyster mushroom mycelium dry weight.
In addition, the invention also provides application of the method for precisely quantifying the bag-cultivated oyster mushroom mycelium biomass in oyster mushroom production.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
the invention uses the phoS gene of oyster mushroom strain as target gene, and establishes absolute fluorescence quantitative PCR standard curve of the phoS gene by fluorescence quantitative PCR. The mixed samples containing different biomass mycelia were prepared by mixing different weights of oyster mushroom mycelia with a constant weight of culture medium 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. Finally, conversion establishes a standard curve between hypha biomass and DNA copy number. The research result of the invention not only provides a good method for accurately quantifying the oyster mushroom mycelia cultivated by clinker or fermentation materials, but also provides a reference for the follow-up research of the relation between the oyster mushroom mycelia biomass and the fruiting body yield and the disclosure of the dynamic change of the oyster mushroom mycelia biomass in different growth and development periods.
Drawings
FIG. 1 PhoS gene gDNA fragment amplification electrophoresis pattern (A) and pMD19T-phoS recombinant plasmid electrophoresis pattern (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 empty plasmid; 2 and 3, pMD19T-phoS recombinant plasmids;
FIG. 2 is a schematic diagram of the design of the phoS gene fluorescent quantitative PCR primer;
FIG. 3 gRTphos-F/gRTphos-R primer annealing temperature screening, M, tans2K Plus II DNA marker;1 to 3, annealing temperature is 53 ℃, 56 ℃,58 ℃ gDNA fragment amplification result; 4 to 6, the annealing temperature is 53 ℃, 56 ℃ and 58 ℃ primer dimer amplification result;
FIG. 4 is a standard curve of absolute fluorescence quantitative PCR for the phoS gene;
FIG. 5A broad scale standard curve between oyster mushroom hypha DNA copy number and biomass;
FIG. 6 is a small scale standard curve between oyster mushroom hypha DNA copy number and biomass;
FIG. 7 accuracy test of oyster mushroom hypha DNA copy number-biomass standard curve;
FIG. 8 application of oyster mushroom hypha DNA copy number-biomass standard curve.
Detailed Description
The invention is further described below in connection with the following detailed description. The present invention is not limited to the following examples.
According to the invention, the phoS gene of the novel 831 strain of oyster mushroom is taken as a target gene, oyster mushroom hypha is taken as a test material, an absolute fluorescence quantitative PCR standard curve of the phoS gene is established by a fluorescence quantitative PCR method, and a linear relation between DNA copy number and oyster mushroom hypha biomass is further established. The experimental result shows that the absolute fluorescence quantitative PCR standard curve of the oyster mushroom phoS gene is y= -2.8917x+34.231, the standard curve (wide range) between oyster mushroom DNA copy number and hypha biomass is y= 0.2973x-0.33347, R 2 Standard curve (small range) between oyster mushroom DNA copy number-hypha biomass of y= 0.1719x-0.0.039, r=0.9987 2 =0.993. The oyster mushroom mycelium biomass has a good linear relation with the DNA copy number, can be used for precisely quantifying the bag-cultivated oyster mushroom mycelium biomass, and is particularly suitable for measuring the oyster mushroom mycelium biomass cultivated by the fermentation material.
Examples
Test materials
Test strains and plasmids
The novel 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% glucose, 0.5% yeast powder, 0.1% KH 2 PO 4 、0.05％MgSO 4 ·7H 2 O、0.01‰V B1 Distilled water 1L. Sterilizing with steam at 115 deg.C for 30 min.
(2) The formula of the pure cotton seed hull culture material comprises the following steps: 100% of cotton seed hulls and 60% of water content.
(3) The formula of the phosphorus-containing culture material comprises the following steps: 70% of cotton seed hulls and 30% of wheat bran are respectively added with phosphorus elements with different concentrations to prepare cultivation matrixes with the phosphorus element contents of 0%, 0.2%, 0.4%, 0.6%, 1.0% and 1.2% respectively, and the water content is 60%. Sterilizing at 100deg.C under normal pressure for 16 hr.
(II) test method
1. Amplification and sequencing of oyster mushroom-specific phoS genes
(1) Primers for amplifying the phoS gene of the novel 831 strain of oyster mushroom were designed according to the P.ostreatus PC15 genome sequence, and the sequences of the primers are as follows:
phoS-F:5'...GGTGATAGTATTGATCGGCGTTATTG...3'
phoS-R:5'...CATGATCGAGAACTTCATGGAAC...3'
(2) The genomic DNA of the new 831 strain of oyster mushroom is used as a template for amplification, and the reaction system is as follows: ddH 2 O34. 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: 98 ℃ for 3min;98℃10s,58℃15s,72℃1min 10s,30 cycles; and at 72℃for 5min.
(3) Performing gel recovery on the PCR product according to the instruction of the Shanghai industrial gel recovery kit;
(4) Performing an enzyme-linked reaction according to the specification of a pMD19-T vector (TAKARA) kit;
(5) E.coli transformation and pMD19T-phoS recombinant plasmid screening were performed according to conventional molecular cloning procedures;
(6) The pMD19T-phoS recombinant plasmid was submitted to large gene sequencing (the sequence of the phoS gene is shown in SEQ ID NO. 1).
2 phoS gene specific fluorescent quantitative PCR amplification primer design and annealing temperature screening
2.1 amplification primer design
According to the primer design principle shown in FIG. 2, according to the sequencing result of the pMD19T-phoS recombinant plasmid, the phoS gene specific real-time fluorescence quantitative PCR amplification primer is designed, wherein the upstream primer gRTphos-F is positioned in an intron, the downstream primer gRTphos-R is positioned in an exon, and the influence caused by the amplification of RNA as a template is eliminated. The primer sequences were as follows:
gRTphoS-F：5’…GTCTCGCCCCTTTATCTGAATG…3’
gRTphoS-R:5’…GGACTTGAACGCTGCGATATTTG…3’
2.2 amplification annealing temperature screening
The reaction system is as follows: ddH 2 O20. Mu.L, 10 XBuffer 2.5. Mu.L, dNTP 2. Mu.L, gRTphos-F0.4. Mu.L, gRTphos-R0.4. Mu.L, gDNA 0.25. Mu.L. The negative control group was not added gDNA.
Setting three annealing temperatures of 53 ℃, 56 ℃ and 58 ℃. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min; denaturation at 94℃for 30 seconds, annealing at 53℃at 56℃at 58℃for 30 seconds, elongation at 72℃for 20 seconds, 35 cycles; incubate at 72℃for 5min. At the end of PCR, 5. Mu.L of the amplified product was subjected to 1.2% agarose gel electrophoresis (FIG. 3).
3. Oyster mushroom mycelium preparation
(1) According to the inoculation amount of 5%, transferring the activated oyster mushroom liquid strain into a GYE liquid culture medium, and carrying out dark shake culture at 25 ℃ and 180rpm for about 5 days.
(2) Mycelium was collected by vacuum filtration and rinsed once with sterile water.
Preparation of 4 oyster mushroom mycelium-culture material mixed sample and extraction of genome DNA
4.1 preparation of oyster mushroom hypha-culture mixture sample
(1) In order to simulate living environment under oyster mushroom hypha cultivation conditions, the invention adopts the steps of uniformly mixing oyster mushroom hypha (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.3, 0.6 and 1.5 g) with pure cotton seed shell culture materials with constant weight (4 g) to prepare hypha-culture material mixed samples, and each treatment is repeated three times. (bag cultivated oyster mushroom hypha has extremely low biomass, not more than 1.5 g.)
(2) The mycelium-culture material mixed sample is added into a mortar precooled by liquid nitrogen, the liquid nitrogen with proper volume is poured into the mortar, and the mixture is rapidly ground to form powder.
(3) Determination of mycelium dry weight: 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 into a 65 ℃ oven for drying to constant weight, and weighing the dry weight of the mycelia.
(4) Determination of pure culture dry weight: accurately weighing 4g of pure cotton seed shell culture material, putting the culture material into a 65 ℃ oven for drying to constant weight, and weighing the dry weight of the pure culture material.
4.2 extraction of oyster mushroom hypha genomic DNA
The invention adopts a CTAB method to extract DNA of a mycelium-culture material mixed sample.
(1) Preparation of the solution
1) 2 XCTAB 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, and stored at room temperature after sterilization.
3) TE buffer:10mM Tris-HCl,1mM EDTA (pH 8.0), and after preparation, sterilized in a vertical autoclave at 121℃for 20min, and stored at room temperature.
4) TE buffer (containing RNase A1): mu.L of RNase A1 (50 mg/mL) was taken into 1mL of TE buffer, and the final concentration of RNase A1 was 1mg/mL. Preserving in a refrigerator at-20 ℃.
5) 3M sodium acetate solution (pH 5.2) (100 mL): 24.61g of anhydrous sodium acetate is weighed, 80mL of ultrapure water is added, the pH is regulated to 5.2 by glacial acetic acid, the volume is fixed to 100mL, sterilization is carried out for 20min at 121 ℃, and the mixture is preserved at room temperature.
6) 70% ethanol (200 mL): 140mL of absolute ethanol (analytically pure) is measured and added into a reagent bottle, 60mL of sterilized ultrapure water is added, and the mixture is stored in a refrigerator at 4 ℃ for standby.
(2) Extraction of oyster mushroom hypha genome DNA
1) Accurately weigh 0.2g of the sample ground to powder with liquid nitrogen into a 2mL centrifuge tube pre-cooled with liquid nitrogen.
2) 400 mu L Soil DNA extraction buffer buffer was added and mixed well with vigorous inversion and vortex.
3) 400mL of 2 XCTAB buffer solution is added, and the mixture is vigorously reversed and mixed by vortex oscillation; the cells were allowed to stand at 55℃for 5min to allow sufficient lysis.
4) Adding 500 mu L of a solution of phenol, chloroform and isoamyl alcohol (25:24:1), and mixing by violent inversion and vortex oscillation; the mixture was centrifuged at 12,000rpm and 4℃for 10min.
5) Transferring about 650 mu L of supernatant to a 1.5mL centrifuge tube by using a pipette; adding 455 mu L of isopropanol, reversing and uniformly mixing to precipitate DNA; the mixture was centrifuged at 12,000rpm at 4℃for 10min, and the supernatant was discarded.
6) Adding 1mL of precooled 70% ethanol, and carrying out vortex vibration; centrifuging at 12,000rpm and 4 ℃ for 10min, and discarding supernatant; placing in a fume hood for 3-5 min to remove the alcohol.
7) Adding 50 mu L of preheated TE buffer (containing RNase A1) to dissolve DNA precipitate, incubating at 65 ℃ for 10min, and inactivating DNase; incubating at 37℃for 30min to remove RNA in the DNA sample.
8) 5. Mu.L of 3M sodium acetate buffer (pH 5.2) and 125. Mu.L of absolute ethanol were added, and the mixture was left in a refrigerator at-80℃for 30 minutes; centrifuging at 12,000rpm and 4 ℃ for 10min, and discarding supernatant;
9) Repeating step 6);
10 50. Mu.L of TE buffer preheated at 65℃was added, and the pellet was dissolved in the buffer by flicking and stored in a refrigerator at-80 ℃.
5 establishment of standard curve between oyster mushroom hypha DNA copy number and biomass
5.1 Construction of absolute fluorescence quantitative PCR standard curve of phoS gene
(1) Extracting pMD19T-phoS plasmid by using a small amount of Shanghai working medium particle extraction kit, and measuring the plasmid concentration;
(2) The extracted pMD19T-phoS plasmid was subjected to 10-fold gradient dilution (10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 )；
(3) Fluorescent quantitative PCR amplification was performed using the diluted pMD19T-phoS plasmid as template and gRTphos-F and gRTphos-R primers, and each sample was repeated three times.
The reaction system is as follows: 2 XSYB Green 10. Mu.L, pMD19T-phoS plasmid at different dilutions 0.5. Mu.L, gRTphos-F0.4. Mu.L, gRTphos-R0.4. Mu.L, ddH 2 O 9μL。
The reaction conditions are as follows: 95 ℃ for 2min;95 ℃ 20s, 56 ℃ 20s, 72 ℃ 20s,40 cycles; 95 ℃ for 5s,4.4 ℃/s; 1min at 65 ℃ and 2.2 ℃/s;65 ℃ to 95 ℃ continuous,0.1 ℃/s; cooling at 40 ℃,2.2 ℃/s.
(4) preparation of PhoS Gene Standard Curve
The copy number of pMD19T-phoS plasmid was calculated for each dilution, and then a standard curve of the phoS gene was drawn with the log10 (copy number) as the abscissa and the Ct value as the ordinate (FIG. 4).
5.2 construction of oyster mushroom hypha DNA copy number-biomass Standard Curve
(1) Fluorescent quantitative PCR amplification was performed using 1. Mu.L of sample DNA as a template and gRTphos-F/gRTphos-R primers, and each sample was repeated three times.
(2) Substituting Ct values corresponding to the samples into a phoS gene absolute fluorescence quantitative standard curve, and calculating log10 (copy number) values of the phoS genes of the samples; the copy number of the phoS gene in each sample was further calculated. To eliminate the difference in moisture content of hyphae from different samples, the present invention characterizes the hyphae biomass in terms of hyphae dry weight. DNA copy number-oyster mushroom hypha biomass standard curve was drawn with hypha dry weight as abscissa and DNA copy number as ordinate (FIGS. 5 and 6).
6 accuracy test of standard curve between oyster mushroom hypha DNA copy number and biomass
Accurately weighing 0g (T1), 0.4g (T2), 1g (T3), 2g (T4) oyster mushroom mycelium and 4g culture material, uniformly mixing to prepare oyster mushroom fungus chaff, weighing 0.2g culture material after uniformly mixing, and extracting genome DNA. Fluorescent quantitative PCR amplification was performed as described above. The Ct value of the phoS gene of each sample was sequentially substituted into the phoS gene standard curve to calculate the copy number of the phoS gene, and further the copy number of the phoS gene was substituted into the DNA copy number-mycelium biomass standard curve to calculate the dry weight of oyster mushroom mycelium in oyster mushroom fungus chaff, which was compared with the actual dry weight of oyster mushroom mycelium (FIG. 7).
The positive correlation exists between the dry weight of oyster mushroom mycelia and the dry weight of actual oyster mushroom mycelia, which is calculated by using the absolute fluorescence quantitative standard curve of the phoS gene and the oyster mushroom mycelia DNA copy number-biomass standard curve, so that the method for accurately quantifying the biomass of the bag-cultivated oyster mushroom mycelia, which is established by the invention, has high accuracy and can be widely applied to oyster mushroom production. Compared with the traditional methods for measuring the concentration of hypha DNA, the protein concentration and the like, the oyster mushroom hypha quantitative method established by the invention can eliminate the interference of the culture material DNA and other microorganism DNA, and can accurately quantify the biomass of oyster mushroom hypha.
Application of 7-oyster mushroom hypha DNA copy number and biomass standard curve
(1) Bag making
1) Accurately weighing cotton seed hulls and 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 dissolution;
3) Adding the dissolved phosphate solution into the mixed sample of cotton seed hulls and wheat bran, and stirring uniformly;
4) Filling the culture material into a 22cm multiplied by 36cm multiplied by 0.004cm polypropylene corner bag, and sealing the sleeve ring;
5) Sterilizing at 100 ℃ under normal pressure for 16 hours;
6) After the material bag is cooled, under aseptic condition, one end is connected with oyster mushroom solid strain, and the strain is covered;
7) The fungus bags are carried to a culture room at 25 ℃ and cultured until hyphae of a certain treatment group grow to half bags.
(2) Qualitative analysis of hypha growth vigor
The fungus bag is opened at a position 1cm away from the tip of the mycelium, and the cross section mycelium growth vigor is recorded by photographing.
(3) Hypha genome DNA extraction
1) Crushing the culture material with mycelium ends, and randomly weighing 5g;
2) Quick freezing with liquid nitrogen, and grinding into powder;
3) Accurately weighing 0.2g of powder into a centrifuge tube, and extracting genome DNA by using a CTAB method.
(4) Quantification of oyster mushroom hypha biomass in culture medium
1) Determining the Ct value of the phoS gene in each sample using the gRTphos-F/gRTphos-R primer;
2) The Ct value is carried into the absolute fluorescence quantitative standard curve of the phoS gene, and log10 (copy number) is calculated;
3) Further calculating the DNA copy number;
4) Substituting the DNA copy number into a oyster mushroom mycelium DNA copy number-biomass standard curve, and calculating to obtain oyster mushroom mycelium dry weight;
5) The dry weight ratio of the culture material is calculated: accurately weighing 100g of the culture material, putting the culture material into a 65 ℃ oven for drying to constant weight, weighing the dry weight of the culture material, and calculating the dry weight ratio;
6) The mycelium biomass of each treated group of oyster mushrooms was normalized by the dry weight of the culture medium to eliminate errors caused by the difference in water content of the culture medium, and finally the dry weight (mg/g dry material) of the mycelium was obtained (fig. 8).
The positive correlation exists between the dry weight of the oyster mushroom mycelia obtained by calculation by using the absolute fluorescence quantitative standard curve of the phoS gene and the DNA copy number-biomass standard curve of the oyster mushroom mycelia and the qualitative observation of the growth vigor of the oyster mushroom mycelia, so that the method for accurately and quantitatively bag-planting the oyster mushroom mycelia, which is established by the invention, has high accuracy and can be widely applied to oyster mushroom production. Compared with the traditional methods for measuring the concentration of hypha DNA, the protein concentration and the like, the oyster mushroom hypha quantitative method established by the invention can eliminate the interference of the culture material DNA and other microorganism DNA, and can accurately quantify the biomass of oyster mushroom hypha.
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 of the oyster mushroom mycelia and the biomass, and can select a proper standard curve to calculate the oyster mushroom mycelia biomass according to the actually measured DNA copy number.
Sequence listing
<110> Henan agricultural university
<120> a method for precisely quantifying the biomass of mycelium of bag-cultivated oyster mushroom 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 (6)
1. The method for quantitatively detecting the biomass of the bag-cultivated oyster mushroom hyphae is characterized by comprising the following steps of:
(1) Oyster mushroom specific phoS gene amplification and sequencing;
(2) The design of a phoS gene fluorescent quantitative PCR primer and optimization of amplification conditions;
(3) Preparing oyster mushroom mycelia;
(4) Preparing an oyster mushroom mycelium-culture material mixed sample and extracting genome DNA;
(5) Constructing an oyster mushroom hypha DNA copy number-biomass standard curve;
(6) Quantifying biomass of oyster mushroom hyphae in the culture material;
the step (1) specifically comprises the following steps:
(1) designing a primer to amplify oyster mushroom specific phoS gene segment;
(2) constructing a pMD19T-phoS recombinant plasmid;
(3) sequencing the pMD19T-phoS recombinant plasmid, wherein the sequence of the phoS gene is shown as SEQ ID NO. 1;
in the step (2), the design principle of the fluorescence quantitative PCR primer of the phoS gene is that an upstream primer gRTphos-F is positioned in an intron, and a downstream primer gRTphos-R is positioned in an exon; the upstream primer gRTphos-F has a sequence of 5'… GTCTCGCCCCTTTATCTGAATG … 3', and the downstream primer gRTphos-R has a sequence of 5'… GGACTTGAACGCTGCGATATTTG …';
the step (5) specifically comprises the following steps:
(1) construction of absolute fluorescence quantitative PCR standard curve of phoS gene
Taking the pMD19T-phoS plasmid constructed in the step (1) as a standard plasmid, measuring the Ct value of the phoS gene at each dilution by real-time fluorescence quantitative PCR, and establishing a standard curve between the copy number and the Ct value;
(2) construction of oyster mushroom hypha DNA copy number-biomass standard curve
Taking the genomic DNA of each sample extracted in the step (4) as a template, measuring the Ct value of the phoS gene in each sample by using gRTphoS-F/gRTphoS-R primers, taking 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 oyster mushroom hypha and biomass, wherein the hypha biomass is characterized by hypha dry weight; drawing a DNA copy number oyster mushroom hypha biomass standard curve by taking the hypha dry weight as an abscissa and the DNA copy number as an ordinate;
the step (6) comprises: determining the Ct value of the phoS gene in each sample by using the gRTphos-F/gRTphos-R primer, substituting the Ct value into a log10 (copy number) -Ct value standard curve, calculating to obtain log10 (copy number), and further calculating the DNA copy number; substituting the DNA copy number into a oyster mushroom mycelium DNA copy number-biomass standard curve, and calculating to obtain the oyster mushroom mycelium dry weight.
2. The method for quantitatively detecting the biomass of bag-cultivated oyster mushroom mycelia according to claim 1, wherein the annealing temperature of the gRTphos-F/gRTphos-R primer in the step (2) is 53-58 ℃.
3. The method for quantitatively detecting the biomass of bag-cultivated oyster mushroom mycelia according to claim 2, wherein the annealing temperature of the gRTphos-F/gRTphos-R primer in the step (2) is 58 ℃.
4. The method for quantitatively detecting the biomass of the mycelium of bag-cultivated oyster mushrooms according to claim 1, wherein the step (4) specifically comprises the following steps: and uniformly mixing oyster mushroom mycelia with different wet weights and culture materials with constant weights, preparing a mycelia-culture material mixed sample, rapidly grinding the mixture into powder under the condition of liquid nitrogen, and extracting genome DNA of the mixed sample by using a CTAB method.
5. The method for quantitatively detecting the biomass of bag-cultivated oyster mushroom mycelia according to claim 4, wherein the step (1) comprises the steps of: a. extracting the pMD19T-phoS plasmid and determining the plasmid concentration; b. performing 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. performing fluorescent quantitative PCR amplification by using the diluted pMD19T-phoS plasmid as a template and using gRTphos-F/gRTphos-R primers, and determining Ct values of samples of each dilution, wherein each sample is repeated three times; d. a standard curve between copy number and Ct value is established.
6. Use of the method for quantitatively detecting bag-cultivated oyster mushroom mycelium biomass according to any one of claims 1-5 in oyster mushroom production.
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| "Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR";Raúl Castanera et al.;《Applied and Environmental Microbiology》;第81卷(第12期);第4120-4129页 * |
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