CN113322309A - Method for quantifying microbial environment change response force - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to the technical field of environmental science and engineering, and discloses a method for quantifying the response force of microbial environmental change. The method comprises the following steps: respectively arranging an isotope labeling group and an unlabeled control group corresponding to the isotope labeling group on a soil sample, wherein the isotope labeling group contains at least one substrate corresponding to a species element, the species element in one substrate exists in an isotope form, and the species performance of each species under different environmental conditions is obtained by using a stable isotope nucleic acid probe technology; selecting a certain species as a target species A, setting a reference environmental condition a and a processing environmental condition b, and calculating a standard effect value by using the difference of species performances respectively corresponding to the target species A under the two environmental conditions so as to measure the species environmental response force of the target species A under the environment with the change of the processing environmental condition b. The method can realize the quantitative analysis of the functional response force of the microorganism species under the environmental change.
Description
Technical Field
The invention relates to the technical field of environmental science and engineering, in particular to a method for quantifying the response force of microbial environmental change.
Background
The physiological metabolic function of a particular microorganism is the result of long-term interactions with other organisms and the natural environment. The molecular regulation mechanism of important physiological processes of microorganisms in a complex environment is researched at a community level, and the mechanism, the driving force and the possible evolution direction of microbial diversity formation can be accurately known. Compared with a pure culture system, the number of microorganisms in the natural environment is huge and the variety is large, most species lack pure culture, the physiological characteristics of the species are greatly prevented from being obtained by people, and therefore the functional function of the species in an ecological system cannot be known. There is a great difficulty in understanding the molecular mechanisms of the physiological metabolic processes of microbial communities in complex environments on an overall level. Therefore, how to quantify the response of the natural environment or artificially assembled microorganism performance to environmental changes at the microbial species level is a very challenging technical challenge.
In order to overcome the gap between microbial species and function, a stable isotope labeling-based method is used, by means of a stable isotope (usually a stable isotope)13C and18o) labeling target substrates, and measuring and calculating the activities of the growth and assimilation metabolic substrates of each species by using density gradient centrifugation in combination with real-time fluorescent quantitative PCR and high throughput sequencing techniques, which is one of the most effective methods for directly linking the microbial population in the natural environment to the microbial process at the species level at present. However, under different spatial and temporal conditions, no relevant research report is found on how the performance of the microbial species responds to environmental changes, including the strength and the response direction of response force, how to perform quantitative analysis on the response force, and the like.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for quantifying the response force of the environmental change of microorganisms, which can realize the quantitative analysis of the functional response force of microorganism species under the environmental change.
In order to achieve the above object, the present invention provides a method for quantifying a response force of a microbial environmental change, comprising the steps of:
s1, respectively arranging isotope labeling groups and unlabeled control groups corresponding to the isotope labeling groups on the soil samples, wherein the isotope labeling groups comprise at least one substrate corresponding to a species element, the species element in one substrate exists in an isotope form, and the species performance of each species under different environmental conditions is obtained by using a stable isotope nucleic acid probe technology;
s2, selecting one species as a target species A, setting a contrast environmental condition a and a processing environmental condition b, and calculating a standard effect value by using the difference of species performances respectively corresponding to the target species A under the two environmental conditions so as to measure the species environmental response force of the target species A under the environment of changing the processing environmental condition b;
wherein the species environmental response force comprises an environmental response intensity and an environmental response direction of the target species A.
Preferably, the treatment environmental condition b is additionally variable with respect to the control environmental condition a by the content of at least one element of the species and/or at least one environmental physical condition.
Preferably, in step S1, the species element is C, O or N.
Preferably, in step S1, the isotope labeling group is set to H2 18A mark group of O,13C-glucose + H2 16A mark group of O,12C-glucose + H2 18A mark group of O,13C-glucose + H2 16O+NH4NO3Marker set and12c-glucose + H2 18O+NH4NO3A labeled group, the unlabeled reference group being set to H2 16O control group,12C-glucose + H2 16O control group and12c-glucose + H2 16O+NH4NO3And (4) a control group.
Preferably, in step S1, the species property includes13Atomic percent of C is greater than and/or equal to18The atomic percent of O is over.
Preferably, in step S1, the process of obtaining species properties of the species corresponding to each of the species elements under different environmental conditions by using the stable isotope nucleic acid probe technology includes:
(1) after the isotope labeling group and the unlabeled control group are cultured, respectively collecting culture samples to perform DNA extraction and density gradient centrifugation to obtain a plurality of buoyancy density layers, and then performing real-time fluorescence quantitative PCR and high-throughput sequencing on DNA of each buoyancy density layer of each culture sample to obtain the bacterial community structure and the number of each buoyancy density layer of each culture sample;
(2) calculating and obtaining the bacterial community structure and the number of the buoyancy density layers corresponding to the isotope labeling group and the unlabeled contrast group respectively13C and18the atomic percent of O is over.
Preferably, the13C and18the calculation formula of the atomic percent of O is as follows:
ΔD＝DLAB–DLIGHT，
WHEAVYMAX＝12.07747+WLIGHT，
wherein, APE is atom percent, WLIGHTIs the average molecular mass of single-stranded DNA of O or C in an unlabeled control group, WLABIs the average molecular mass of the single-stranded DNA of O or C in the isotopically labeled group, DLIGHTDensity of O or C in unlabeled control group, DLABIs the density of O or C in the isotopically labeled group, and Δ D is the cause of O or C18O or13Density difference of C generation, WHEAVYMAXRespectively, is theoretically O or C in DNA18O or13Maximum molecular mass at C mark, K is18O and13background fractional abundance of C, 0.002000429 and 0.01111233, respectively.
Preferably, in step S2, the standard effect value is calculated by the formula:
df＝nt+nc–2，
wherein ES is a standard effect value,
the average atomic percentage of the target species a corresponding to the processing environmental condition b is exceeded,
the average atomic percentage corresponding to the target species A is over when the target species A is compared with the environmental condition a, gamma is gamma function, df is freedom degree, ntNumber of samples to deal with environmental condition b, ncNumber of samples to control environmental condition a, SpooledIs the combined standard deviation of the atomic percent excesses of target species a at treatment ambient condition b and control ambient condition a.
Preferably, the expression of the community level corresponding to the target species a is as follows:
wherein WM is the weighted average of communities, piIs the relative abundance of said target species a in the population; functioniIs the atomic percent of the target species A in the community exceeds APE or standard effect value ES.
Through the technical scheme, the invention has the beneficial effects that:
the method provided by the invention is based on a quantitative stable isotope probe technology, the species performance of each species under different environmental conditions is obtained, then the reference environmental condition a is taken as a reference, the species performance change of the reference environmental condition a and the processing environmental condition b under two environmental conditions is compared, and a standard effect value is calculated, so that the species environmental response stress of a target species A under the change of the processing environmental condition b is measured, wherein the species environmental response stress comprises species environmental response intensity and species environmental response direction; the method provided by the invention considers the information of species functional traits, can overcome the technical problem that species environmental responsiveness of natural environment or artificially assembled microorganisms cannot be quantified, does not need to perform species isolation culture of microorganisms based on the traditional method, only needs to perform stable isotope labeling on an in-situ environment sample, performs quantitative calculation by combining a molecular biology technology, and develops and clarifies the research idea of exploring microbial community function maintenance under environmental change from the perspective of species functional traits.
Further advantages of the present invention, as well as the technical effects of preferred embodiments, are further described in the following detailed description.
Drawings
FIG. 1 is a block diagram of a method for quantifying the response of a change in microbial environment according to an embodiment of the present invention;
FIG. 2 is a flow chart of the stable isotope nucleic acid probe technique provided by the present invention, wherein qPCR is real-time fluorescence quantitative PCR;
FIG. 3 is a graph of the relationship between the species performance and the soil elevation obtained by the method for quantifying the response force of the microbial environmental change provided by the invention;
FIG. 4 is a graph showing the relationship between the environmental response of species and the elevation of soil obtained by the method for quantifying the environmental response of microorganisms provided by the present invention.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for quantifying the response force of microbial environmental change, which is characterized by comprising the following steps of:
s1, respectively arranging isotope labeling groups and unlabeled control groups corresponding to the isotope labeling groups on the soil samples, wherein the isotope labeling groups comprise at least one substrate corresponding to a species element, the species element in one substrate exists in an isotope form, and the species performance of each species under different environmental conditions is obtained by using a stable isotope nucleic acid probe technology;
s2, selecting one species as a target species A, setting a contrast environmental condition a and a processing environmental condition b, and calculating a standard effect value by using the difference of species performances respectively corresponding to the target species A under the two environmental conditions so as to measure the species environmental response force of the target species A under the environment of changing the processing environmental condition b;
wherein the species environmental response force comprises an environmental response intensity and an environmental response direction of the target species A.
According to the invention, the isotope labeling group and the unlabeled control group are correspondingly arranged, specifically, the isotope labeling group and the unlabeled control group are respectively added with substrates containing species elements of the same kind and the same quantity in the original soil sample, so that the species elements and the content of the substrate are the same, and in the isotope labeling group, the species elements in one substrate exist in a corresponding isotope form, so that the species performance of the species under various environmental conditions is obtained by using the change labeling of the isotope elements. The added substrate in the original soil sample can be one or more, and can be designed and selected according to the species to be analyzed. In the present invention, the environmental conditions may be a variety of environmental factors affecting the soil environment, including but not limited to chemical factors, physical factors, such as the content of various species elements, temperature, humidity (i.e., H may be used)2Measured by O content), ph, and the like.
It should be noted that the species element and the substrate are in one-to-one correspondence, and if other substrates added at the same time also contain the species element, the species element exists only in the form of an isotope in the corresponding substrate, and exists in the form of a conventional isotope in the other substrates. For example, the substrate glucose corresponds to the species element C, which can be used in the corresponding isotopic label set13C-glucose as a labelled substrate, substrate H2O is O, and can be used in corresponding isotope labeling groups18O-H2O as a labelled substrate, substrate NH4NO3The corresponding species element is N; in general, H2The addition of O is determined by calculating the water content in the soil, and H is carried out according to the soil water content of 70 percent2And (4) adding O.
Specifically, in step S1, the isotope labeling group may be set to H2 18A mark group of O,13C-glucose + H2 16A mark group of O,12C-glucose + H2 18A mark group of O,13C-glucose + H2 16O+NH4NO3Marker set and12c-glucose + H2 18O+NH4NO3A marker group, and the unmarked control group can be set as H2 16O control group,12C-glucose + H2 16O control group and12c-glucose + H2 16O+NH4NO3And (4) a control group. Wherein H2 18O marker group and H2 16The reference group O corresponds to the reference group,13c-glucose + H2 16O mark group and12c-glucose + H2 16The reference group O corresponds to the reference group,12c-glucose + H2 18O mark group and12c-glucose + H2 16The reference group O corresponds to the reference group,13c-glucose + H2 16O+NH4NO3Marker set and12c-glucose + H2 16O+NH4NO3The control group is corresponded to the control group,12c-glucose + H2 18O+NH4NO3Marker set and12c-glucose + H2 16O+NH4NO3The control group corresponded.
According to the present invention, the environmental factors of the soil include contents of various species elements and physical environmental conditions including soil temperature/humidity/ph and the like. As a specific arrangement mode of the isotope labeling group and the unlabeled control group, the isotope labeling group can also combine the species elements with the physical environmental conditions to examine the species performance of each species when the physical environmental conditions change, and exemplarily, the isotope labeling group is arranged to13C-glucose + H2 16When the group is marked by O,12c-glucose + H2 16The O control group is an unlabeled control group, and the soil temperature/humidity/ph of the two groups are increased or decreased to examine the species performance of each species at different temperatures/humidity/ph.
According to the invention, the environmental response intensity of the target species a specifically refers to the magnitude of the response value of the target species a; the species environmental response direction refers to whether the response value of the target species A is increased or decreased after the target species A is subjected to environmental change.
According to the invention, the species element may be any chemical element, for example H, C, N, O, P or the like. Preferably, in step S1, the species element is C, O or N.
According to the present invention, in step S1, the species property refers to a change rate of the species element corresponding to each species under different environmental conditions, and may be, for example, a growth rate of the species element O corresponding to the species or a carbon assimilation rate of the species element C corresponding to the species. Illustratively, in the present invention, the species property includes13Atomic percent of C is greater than and/or equal to18The atomic percent of O is over.
According to the present invention, stable isotope nucleic acid probe technology can be applied to the method steps already disclosed in the prior art. Preferably, in step S1, the process of obtaining species properties of the species corresponding to each of the species elements under different environmental conditions by using the stable isotope probe nucleic acid probe technology includes:
(1) after the isotope labeling group and the unlabeled control group are cultured, respectively collecting culture samples to perform DNA extraction and density gradient centrifugation to obtain a plurality of buoyancy density layers, and then performing real-time fluorescence quantitative PCR and high-throughput sequencing on DNA of each buoyancy density layer of each culture sample to obtain the bacterial community structure and the number of each buoyancy density layer of each culture sample;
(2) calculating and obtaining the bacterial community structure and the number of the buoyancy density layers corresponding to the isotope labeling group and the unlabeled contrast group respectively13C and/or18The atomic percent of O is over.
Illustratively, the13C and18the specific calculation process for the atomic percent of O is as follows: first, the number of each species in each density layer is calculated according to the bacterial colony structure and the total number of each density layer of each culture sample, and the density of each species in each layer is calculated by combining the total density of each layer, so that the density (D) of each species in the sample corresponding to the unlabeled control group can be obtainedLIGHT) Density in a sample corresponding to the isotopic label set (D)LAB) And density difference (Δ D ═ D) due to labelingLAB–DLIGHT) Further, the formula is calculated as follows18O and13atomic percent of C over (APE):
WHEAVYMAX＝12.07747+WLIGHT，
wherein, APE is atom percent, WLIGHTIs the average molecular mass of single-stranded DNA of O or C in an unlabeled control group, WLABIs the average molecular mass of the single-stranded DNA of O or C in the isotopically labeled group, DLIGHTDensity of O or C in unlabeled control group, DLABIs the density of O or C in the isotopically labeled group, and Δ D is the cause of O or C18O or13Density difference of C generation, WHEAVYMAXRespectively, is theoretically O or C in DNA18O or13Maximum molecular mass at C mark, K is18O and13background fractional abundance of C, 0.002000429 and 0.01111233, respectively.
According to the invention, the content of at least one species element and/or at least one environmental physical condition may be present as variables in the control environmental condition a, and the content of at least one said species element and/or at least one environmental physical condition is added as a variable to the treatment environmental condition b with respect to the control environmental condition a. The variable environmental factors added for the processing environmental condition b are different from the original variable environmental factors of the comparison environmental condition a.
Illustratively, the control environmental condition a is the addition of a species element O, and the treatment environmental condition b is the addition of a species element O and C, i.e. a standard effect value is calculated by the material variable when the target species a changes in both additions at C, O and the material variable when only O is added, so as to measure the species environmental response force of the target species a in the environment of the change of the addition C; alternatively, the control environmental condition a is the addition of the species element C, and the processing environmental condition b is the addition of the species element C and the temperature (belonging to the environmental physical condition) is increased, that is, the standard effect value is calculated by the material variable of the target species a when the addition of C and the temperature increase are changed and the material variable when only the addition of C is changed, so as to measure the species environmental response of the target species a under the environment with the temperature increase.
According to the invention, in step S2, the standard effect value is calculated by the formula:
df＝nt+nc–2，
wherein ES is a standard effect value,
the average atomic percentage of the target species a corresponding to the processing environmental condition b is exceeded,
the average atomic percentage corresponding to the target species A is over when the target species A is compared with the environmental condition a, gamma is gamma function, df is freedom degree, ntNumber of samples to deal with environmental condition b, ncNumber of samples to control environmental condition a, SpooledIs the combined standard deviation of the atomic percent excesses of target species a at treatment ambient condition b and control ambient condition a.
According to the invention, the species performance and species environmental response of the target species a can be extended to a community level, i.e. a weighted average (WM) of communities, the expression of the community level corresponding to the target species a being:
wherein WM is the weighted average of communities, piIs the relative abundance of said target species a in the population; functioniIs the atomic percent of the target species A in the community exceeds APE or standard effect value ES.
The present invention will be described in detail below by way of examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. In the following examples, soil samples were taken from the mountain area in the middle of the lokia mountain, and other materials, reagents and the like used therein were commercially available without specific reference.
Example 1
Taking soil samples with four altitude gradients (1750m, 2000m, 2250m and 2500m) as an example, 12 samples (including 3 repetitions) are collected from low to high altitude for subsequent labeling experiments, and the labeling experiment of each sample is implemented by the following steps:
step 1, by adding a labeled substrate (13C-glucose and18O-H2o) and conventional substrates: (12C-glucose, H2 16O、NH4NO3) Carrying out a soil marking experiment, and setting eight treatment groups in total, specifically: h2 18A group of O marks (18O; b),13C-glucose + H2 16O mark group (13C; g),12C-glucose + H2 18O flag group (12C-18O; d),13C-glucose + H2 16O+NH4NO3A set of flags (13C _ N; h),12C-glucose + H2 18O+NH4NO3A set of flags (12C _18O _ N; f); h2 16O a reference group (16O; a),12C-glucose + H2 16O a control group (12C; C),12C-glucose + H2 16O+NH4NO3Control group (12C _ N; e), see FIG. 1;
adding 1g dry weight of soil samples corresponding to the eight treatment groups into a 15ml tube, keeping the water content of the soil at 70%, placing the tube in a dark condition at room temperature for pre-culture for one week, and after the pre-culture is finished, adding corresponding substrates according to the settings of the eight treatment groups in the step 1, wherein the labeled substrates are13C-glucose and18O-H2the addition amounts of O, glucose and nitrogen were 1000. mu. g C/g dry weight of soil and 100. mu. g N/g dry weight of soil, H2The addition of O is determined by calculating the water content in the soil, and H is carried out according to the soil water content of 70 percent2O or18O-H2Adding O; culturing for one week after adding substrate, collecting culture sample, performing DNA extraction and density gradient centrifugation, performing real-time fluorescence quantitative PCR and bacteria 16S rRNA gene high-throughput sequencing on DNA of each density layerObtaining the bacterial community structure and the quantity of each density layer, and the specific process is shown in figure 2;
wherein, the extraction of the DNA of the culture sample adopts a commercial kit for extraction, the concrete steps refer to the instruction of a FastDNA SPIN kit (Vista, USA), and the concrete process of DNA density gradient centrifugation is as follows: adding about 1 mu g of DNA, 600ml of buffer solution and 2.6ml of cesium chloride solution into a centrifuge tube, centrifuging for 36h at the rotating speed of 45400rpm and the temperature of 20 ℃, so as to divide the centrifugate into 16-18 density layers, measuring by using a refractive index meter to obtain the buoyancy density of each layer of DNA, and performing real-time fluorescence quantitative PCR and bacterial 16S rRNA gene high-throughput sequencing by adopting primers 515F and 806R:
nucleotide sequence of primer 515F: 5 '-GTGCCAGCMGCCGCGGTAA-3';
nucleotide sequence of primer 806R: 5 '-GGACTACVSGGGTATCTAAT-3';
the specific calculation process is as follows: first, the number of each species in each density layer is calculated according to the bacterial colony structure and the total number of each density layer of each culture sample, and the density of each species in each layer is calculated by combining the total density of each layer, so that the density (D) of each species in the sample corresponding to the unlabeled control group can be obtainedLIGHT) Density in a sample corresponding to the isotopic label set (D)LAB) And density difference (Δ D ═ D) due to labelingLAB–DLIGHT) And can thus be calculated18O and13atomic Percent Excess (APE) of C, expressed as follows:
WHEAVYMAX＝12.07747+WLIGHT，
wherein, APE is atom percent, WLIGHTIs the average molecular mass of single-stranded DNA of O or C in an unlabeled control group, WLABIs the average molecular mass of the single-stranded DNA of O or C in the isotopically labeled group, DLIGHTDensity of O or C in unlabeled control group, DLABIs the density of O or C in the isotopically labeled group, and Δ D is the cause of O or C18O or13Density difference of C generation, WHEAVYMAXRespectively, is theoretically O or C in DNA18O or13Maximum molecular mass at C mark, K is18O and13background fractional abundance of C, 0.002000429 and 0.01111233, respectively;
species properties of the target species a in the above 5 groups of "unlabeled control group vs. isotope labeled group" (i.e. under 5 environmental conditions) were calculated, and the results are shown in fig. 3; the species performance of each species is expanded to the community level to obtain a weighted average (WM) of the community, and fig. 3 further shows the relationship between the community weighted average and the altitude gradient of the species performance.
referring to fig. 1, the responsiveness of the growth rate of the target species a after glucose addition is obtained with the humidity of the soil as a control environmental condition a, the addition of the species element C and the humidity as a treatment environmental condition b, i.e., with the growth rate of the target species a without the addition of a carbon and nitrogen substrate as a control group and the growth rate of the target species a with the addition of glucose as a treatment group [ growth rate (added carbon) vs. growth rate (not added) ]; or taking the humidity of the soil as a control environmental condition a, the addition of the species element C + N and the humidity as a processing environmental condition b, namely taking the growth rate of the target species A under the condition of not adding a carbon and nitrogen substrate as a control group, and the growth rate of the target species A under the condition of adding glucose and nitrogen as a processing group [ growth rate (adding carbon and nitrogen) vs. growth rate (not adding) ], so as to obtain the responsiveness of the growth rate of the target species A after adding glucose and nitrogen; or the addition of the species element C is taken as a control environmental condition a, the addition of the species elements N and C is taken as a processing environmental condition b, namely the growth rate of the target species A under the condition of adding glucose is taken as a control group, the growth rate of the target species A under the condition of adding glucose and nitrogen is taken as a processing group [ growth rate (adding carbon and nitrogen) vs. growth rate (adding carbon), the responsiveness of the growth rate of the target species A after the nitrogen is added is obtained;
the environmental response of the species, i.e., the standard effect value (ES), is expressed as follows:
df＝nt+nc–2，
wherein ES is a standard effect value,
the average atomic percentage of the target species a corresponding to the processing environmental condition b is exceeded,
the average atomic percentage corresponding to the target species A is over when the target species A is compared with the environmental condition a, gamma is gamma function, df is freedom degree, ntNumber of samples to deal with environmental condition b, ncNumber of samples to control environmental condition a, SpooledIs the combined standard deviation of the atomic percent excesses of target species a at treatment ambient condition b and control ambient condition a.
According to the method of the present invention, the species environmental response of the target species a under the above 4 environmental changes is calculated, the species performance of the target species a is expanded to the community level, and a weighted average (WM) of the community is obtained, and the result is shown in fig. 4 to further show the altitude gradient pattern of the community weighted average of the species environmental response of the target species a.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. A method for quantifying the response force of microbial environmental change is characterized by comprising the following steps:
s1, respectively arranging isotope labeling groups and unlabeled control groups corresponding to the isotope labeling groups on the soil samples, wherein the isotope labeling groups comprise at least one substrate corresponding to a species element, the species element in one substrate exists in an isotope form, and the species performance of each species under different environmental conditions is obtained by using a stable isotope nucleic acid probe technology;
s2, selecting one species as a target species A, setting a contrast environmental condition a and a processing environmental condition b, and calculating a standard effect value by using the difference of species performances respectively corresponding to the target species A under the two environmental conditions so as to measure the species environmental response force of the target species A under the environment of changing the processing environmental condition b;
wherein the species environmental response force comprises an environmental response intensity and an environmental response direction of the target species A.
2. The quantification method according to claim 1, wherein the treatment environmental condition b is additionally variable with respect to the control environmental condition a by the content of at least one element of the species and/or at least one environmental physical condition.
3. The quantification method according to claim 1 or 2, wherein in step S1, the species element is C, O or N.
4. The quantitative method of claim 3, wherein in step S1, the isotopic marker set is set to H2 18A mark group of O,13C-glucose + H2 16A mark group of O,12C-glucose + H2 18A mark group of O,13C-glucose + H2 16O+NH4NO3Marker set and12c-glucose + H2 18O+NH4NO3A labeled group, the unlabeled reference group being set to H2 16O control group,12C-glucose + H2 16O control group and12c-glucose + H2 16O+NH4NO3And (4) a control group.
5. The quantification method according to any one of claims 4, wherein the species property comprises the species property in step S113Atomic percent of C is greater than and/or equal to18The atomic percent of O is over.
6. The quantitative method of claim 5, wherein the step S1 of obtaining the species property of the species corresponding to each of the species elements under different environmental conditions by using the stable isotope nucleic acid probe technique comprises:
(1) after the isotope labeling group and the unlabeled control group are cultured, respectively collecting culture samples to perform DNA extraction and density gradient centrifugation to obtain a plurality of buoyancy density layers, and then performing real-time fluorescence quantitative PCR and high-throughput sequencing on DNA of each buoyancy density layer of each culture sample to obtain the bacterial community structure and the number of each buoyancy density layer of each culture sample;
(2) calculating and obtaining the bacterial community structure and the number of the buoyancy density layers corresponding to the isotope labeling group and the unlabeled control group respectively13C and/or18The atomic percent of O is over.
7. The quantification method according to claim 6, wherein the method comprises13C and18the calculation formula of the atomic percent of O is as follows:
ΔD＝DLAB–DLIGHT，
WHEAVYMAX＝12.07747+WLIGHT，
wherein, APE is atom percent, WLIGHTIs the average molecular mass of single-stranded DNA of O or C in an unlabeled control group, WLABSingle-chain of O or C in isotopic labelling groupAverage molecular weight of DNA, DLIGHTDensity of O or C in unlabeled control group, DLABIs the density of O or C in the isotopically labeled group, and Δ D is the cause of O or C18O or13Density difference of C generation, WHEAVYMAXRespectively, is theoretically O or C in DNA18O or13Maximum molecular mass at C mark, K is18O and13background fractional abundance of C, 0.002000429 and 0.01111233, respectively.
8. The quantitative method according to claim 7, wherein in step S2, the standard effect value is calculated by the formula:
df＝nt+nc–2，
wherein ES is a standard effect value,
the average atomic percentage of the target species a corresponding to the processing environmental condition b is exceeded,
the average atomic percentage corresponding to the target species A is over when the target species A is compared with the environmental condition a, gamma is gamma function, df is freedom degree, ntNumber of samples to deal with environmental condition b, ncNumber of samples to control environmental condition a, SpooledIs the combined standard deviation of the atomic percent excesses of target species a at treatment ambient condition b and control ambient condition a.
9. The quantification method according to claim 8, wherein the colony level corresponding to the target species A is expressed as:
wherein WM is the weighted average of communities, piIs the relative abundance of said target species a in the population; functioniIs the atomic percent of the target species A in the community exceeds APE or standard effect value ES.
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