CN103966318A - Method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology - Google Patents
Method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology Download PDFInfo
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Abstract
A method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology comprises the followings steps: (1), collecting a paddy soil sample; (2), performing a micro universe cultivation experiment of <13>C-formic acid; (3), performing centrifugation layering on the microbe total DNA of <13>C-formic acid cultivation soil by using an ultracentrifuge; (4), performing real-time quantitative PCR analysis and finger-print analysis on methanogenic archaea genes in layers of multiple buoyant densities after layering to judge whether the paddy soil contains the formic acid utilization type methanogenic archaea with metabolic activity. Therefore, the method can be used for keenly revealing the paddy field formic acid utilization type methanogenic archaea in situ based on the DNA-based stable isotope probing technology, and has great significance on the understanding of the paddy field nutrient cycling process driven by microbes and the cognition of the ecological functions of paddy field methanogenic archaea functional groups.
Description
Technical field
The present invention relates to one and utilize DNA stability isotope probe (DNA-based Stable Isotope Probing) original position announcement differentiation rice field formic acid to utilize type to produce the method for the ancient bacterium of methane, belong to Ecological Distribution of Soil Microorganisms and learn field.
Background technology
Approximately 1.5 hundred million hectares of whole world rice field areas, 75% in waterflooding state.Anaerobic environment makes rice soil contain abundant small molecular organic acid.By these materials of metabolism, microorganism is driving the biogeochemical cycle process of paddy ecosystem.Formic acid is one of important intermediate in the organic matter degradation process of rice field, and the concentration in rice soil can be up to 150 μ M.In rice soil, multiple-microorganism can metabolism formic acid, carries out sulfate reduction and iron reduction process as multiple anaerobic bacterium in Clostridia guiding principle can utilize formic acid for electron donor.Meanwhile, formic acid is also the important as precursors of paddy methane discharge: annual Estimation accounts for the 4-9% of annual global methane emission, about 25-54 Tg, wherein formic acid and H
2/ CO
2the methane content producing closes the 10-30% that accounts for Estimation.In view of formic acid is the important prerequisite of paddy methane discharge, and the high-content of formic acid in rice soil, we infer the directly ancient bacterium of product methane of metabolism formic acid of certain existence in rice soil.But formic acid utilizes type to produce the ancient bacterium of methane and but there is not yet report in rice soil.Its reason the first, current educable microorganism only accounts for 1% of whole microorganisms, a large amount of microorganisms can only be in specific habitat survival and carry out ecological functions, also cannot pure culture; The second, producing the ancient bacterium of methane is strictly anaerobic microorganism, and it is very high to the requirement of growing environment, and traditional method is difficult to screen; Can utilize formic acid growth although the 3rd partly produces the ancient bacterium of methane under pure culture condition, pure culture condition can not represent the original position habitat in rice soil, so the bacterial strain obtaining can not reflect the truth in rice soil.The DNA stability isotope probe technology (DNA-based Stable Isotope Probing) that developed recently gets up for our situ identification provide may.
DNA stability isotope probe technology is in conjunction with real-time quantitative PCR and finger printing equimolecular biological method, can directedly excavate the Microbial resources that participate in specific ecological process in complicated soil environment, be comparatively effectively one of means of current soil functional microorganism situ identification.DNA stability isotope probe technology ultimate principle is in soil, to add the metabolism substrate that contains stable isotope mark, when specific soil microorganisms utilizes this labeled substrate growth and breeding, on will assimilation substrate containing the element of mark, and for the synthesis of its biomass, as DNA etc.Therefore,, by environment DNA being extracted to isolation identification and compare of analysis, can identify the functional microorganism that drives specific ecological process in soil.The method can accurately disclose the microorganism of carrying out specific function, has become one of important means of situ identification functional microorganism.The importance in biogeochemical cycle process in view of rice field formic acid and the ancient bacterium of product methane, this technology formic acid in rice soil utilizes type to produce the application in the ancient dientification of bacteria of methane, will contribute to expand rice field Nutrient Cycling process that we drive Institute of Micro-biology and rice field and produce the cognition of the ecological functions of the ancient bacterium functional group of methane.
Summary of the invention
the technical problem solving:technical problem to be solved by this invention is to provide a kind of DNA stability isotope probe (DNA-based Stable Isotope Probing) original position announcement differentiation rice field formic acid to utilize type to produce the method for the ancient bacterium of methane, to fill up relevant theory blank, the ecological functions cognition that the ancient bacterium functional group of methane is produced in the rice field Nutrient Cycling process driving for understanding microorganism and rice field has great contribution.
technical scheme:one of the present invention utilizes DNA stability isotope probe (DNA-based Stable Isotope Probing) original position announcement differentiation rice field formic acid to utilize type to produce the method for the ancient bacterium of methane, and its technical process as shown in Figure 1, specifically comprises the following steps:
(1) gather rice soil sample;
(2) carry out
13the microcosm of C-formic acid is cultivated experiment;
(3) utilize ultra-high speed whizzer pair
13the total DNA of soil microorganisms that C-formic acid is cultivated carries out centrifugal layering;
(4) the ancient bacterium gene of the product methane in multiple buoyant densities after layering is carried out to Real-time PCR Analysis and fingerprint map analyzing, finally determine that the formic acid in this rice soil with metabolic activity utilizes type to produce the ancient bacterium of methane.
The step that described microcosm is cultivated experiment is: by 100 mg
13c-formic acid joins and is equipped with in the 120 mL vials that 5 grams of dry ground are heavy, is adjusted to 60% of the maximum water holding weight of field soil with aseptic deionized water, and by use rubber stopper seal after air in nitrogen replacement vial, to keep the anaerobic environment of culture system; Simultaneously with
12c-formic acid and do not add being treated to of formic acid and be respectively contrast, places 28 DEG C of incubator constant temperature culture, extracts the upper layer of air of 1.2 mL every 3 days out, utilizes gas chromatograph for determination concentration of methane gas to change, and treats that methane concentration starts to reduce to stop cultivating experiment.
The centrifugal stratification step of described ultra-high speed is: after microcosm cultivation experiment stops, extracting test kit extract the total DNA of soil microorganisms with soil DNA; Total the soil microorganisms of 2.0 μ g DNA, Gradient Buffer damping fluid and 1.85 g/mL cesium chloride solutions, by 0.1 mL, 0.9 mL and 4.9 mL volume mixture, are got to ultra-high speed whizzer on 5.1 mL mixed solutions; Centrifugal 44 hours of the centrifugal speed of 45 krpm at 20 DEG C; After centrifugal end, the mixed solution in centrifuge tube is carried out to layering with fixed flow rate pump, every layer of 340 μ L volume, points 15 layers; With PEG6000 and every layer of DNA of 70% washing with alcohol.
The determination methods of described Real-time PCR Analysis and fingerprint map analyzing is: the ancient bacterium Auele Specific Primer of the product methane 1106F/1378R based on as shown in SEQ ID NO.1 and SEQ ID NO.2, utilizes real-time quantitative PCR to analyze the ancient bacterium gene copy number of the product methane in each buoyant density layer; Utilizing PCR-DGGE finger printing and PCA figure to fall to forming to the ancient flora of product methane in buoyant density double-layer and light layer analyzes; Utilize phylogenetic tree analysis to judge and in buoyant density double-layer, produce the ancient fungus kind of methane.
beneficial effect:to sum up, the present invention utilizes type to produce the ancient bacterium of methane by announcement rice field formic acid can be sharp based on DNA stability isotope probe (DNA-based Stable Isotope Probing), original position, and the ecological functions cognition that the ancient bacterium functional group of methane is produced in the rice field Nutrient Cycling process driving for understanding microorganism and rice field has great meaning.
Brief description of the drawings
Fig. 1 is technological line schematic diagram;
Fig. 2 is that in microcosm cultivating process, discharge of methane changes schematic diagram;
Fig. 3 produces the ancient bacterium gene copy number of methane distribution schematic diagram in different buoyant density layers, wherein A is the ancient bacterium 16S rRNA gene copy number of known product methane and Cycle threshold(
c t) value typical curve and linear equation; B is for producing the ancient bacterium 16S of methane rRNA gene copy number, and PCR product is through solubility curve (Melting curve analysis) and agarose electrophoresis checking schematic diagram;
Fig. 4 produces the ancient bacterium 16S of methane rRNA gene copy number graph of a relation in different buoyant density layers;
Fig. 5 produces the finger printing schematic diagram that the ancient flora of methane falls to forming in different buoyant density layers;
Fig. 6 produces the PCA that the ancient flora of methane falls to forming differentiation to analyze schematic diagram in different buoyant density layers;
Fig. 7 is that the phylogenetic tree that produces the ancient bacterium of methane is analyzed schematic diagram.
Embodiment
Below in conjunction with concrete embodiment, technical scheme of the present invention is described in further detail, described embodiment is only for illustrating the present invention instead of restriction the present invention.
embodiment 1
Taking Jiangdu of Yangzhou City rice soil as research object, application DNA stability isotope probe original position discloses formic acid in differentiation rice soil and utilizes type to produce the ancient fungus kind of methane.
(1)
13the microcosm of C-formic acid is cultivated experiment
By 100 mg's
13c-formic acid joins and is equipped with in the 120 mL vials that 5 grams of dry ground are heavy, and aseptic deionized water is adjusted to 60% of the maximum water holding weight of field soil, with after air in nitrogen replacement vial by rubber stopper seal, with
12c-formic acid and do not add formic acid soil incubation and be respectively contrast.Place 28 DEG C of incubator constant temperature culture, extracted the upper layer of air of 1.2 mL every 3 days out, utilize gas chromatograph for determination concentration of methane gas to change (Fig. 2), treat that methane concentration starts reduction and stops cultivating experiment.
(2) the total DNA of soil microorganisms is carried out to the centrifugal layering of ultra-high speed
After microcosm cultivation experiment stops, extracting test kit with soil DNA and extract the total DNA of soil microorganisms; Total the soil microorganisms of 2.0 μ g DNA, Gradient Buffer damping fluid and 1.85 g/mL cesium chloride solutions, by 0.1 mL, 0.9 mL and 4.9 mL volume mixture, are got to ultra-high speed whizzer on 5.1 mL mixed solutions; The centrifugal speed of 45 krpm at 20 DEG C (is about 190000 × g) centrifugal 44 hours; After centrifugal end, the mixed solution in centrifuge tube is carried out to layering with fixed flow rate pump, every layer of 340 μ L volume, points 15 layers; With PEG6000 and every layer of DNA of 70% washing with alcohol.
(3) analysis of the ancient bacterium gene copy number of the product methane in each buoyant density after layering
By after DNA layering, based on producing the ancient bacterium Auele Specific Primer of methane 1106F(5'-TTWAGTCAGGCAACGAGC-3')/1378R(5'-TGTGCAAGGAGCAGGGAC-3'), utilize CFX96
tMthermal Cycler(Bio-Rad) instrument carries out quantitative PCR mensuration to the ancient bacterium 16S of product methane in each buoyant density layer of different treatment rRNA gene fragment, concrete steps are as follows: model produces the typical curve of the ancient bacterium 16S of methane rRNA gene fragment, utilize the amplification of 1106F/1378R primer pair to produce the ancient bacterium reference culture of methane 16S rRNA gene fragment, obtain the PCR product that produces the ancient bacterium 16S of methane rRNA gene fragment, clone test kit with Promega PCR product is carried out to TA clone, select and test sun with PCR and find the clone that contains correct clip size by blue hickie, this clone is cultivated in the liquid LB substratum that contains ammonia benzyl to expansion, utilize Takara MiniBEST Plasmid Purification Kit to extract clone and expand plasmid DNA the purifying in liquid, utilize ultramicron nucleic acid concentration determinator Nanodrop to measure plasmid DNA concentration, according to the molecular weight of plasmid and Avogadro constant (6.02 × 10
23molecule/mol) convert out and in every microlitre plasmid DNA, contain the copy number that produces the ancient bacterium 16S of methane rRNA gene fragment, be diluted to a series of every microlitres 1.0 × 10 with 10 times of dilution methods
3~ 1.0 × 10
8gene copy number standard specimen, utilizes real-time fluorescence quantitative PCR test kit and real-time fluorescence quantitative PCR instrument CFX96
tMthermal Cycler(Bio-Rad) set up the ancient bacterium 16S rRNA gene copy number of known product methane and Cycle threshold(
c t) value typical curve and linear equation (Fig. 3 A), real-time fluorescence quantitative PCR system is according to SYBR
premix Ex Taq tM kit(TaKaRa) test kit explanation.According to this linear equation, utilize external standard method according to environmental sample
c tvalue calculates in environmental sample and produces the ancient bacterium 16S of methane rRNA gene copy number, and PCR product, through solubility curve (Melting curve analysis) and agarose electrophoresis checking (Fig. 3 B), is confirmed its specificity and validity.
As can be seen from Figure 4
13c-formic acid produces the ancient bacterium of methane gene copy number maximum value in processing appears at i.e. 1.735 g/mL of buoyant density double-layer; In contrast,
12c-formic acid produces the ancient bacterium of methane maximum gene copy number in processing appears at the light layer of buoyant density (1.717 g/mL).In addition, adding the ancient bacterium gene copy number of the acid-treated product methane of first maximum value is all greater than and does not add formic acid processing.In this presentation of results Some Paddy Soil, have the ancient bacterium of the methane of producing to utilize formic acid growth, thereby its gene copy number increase, in addition
13c-formic acid has had the ancient bacterium metabolism of the methane of producing in processing
13c-formic acid, thus himself DNA " becomes heavy ", increases in the quantity of buoyant density double-layer.
(4) the PCR-DGGE fingerprint map analyzing that after layering, the ancient flora of the product methane in multiple buoyant density layers falls to forming
Subsequently, right
13c-formic acid is processed the light layer of buoyant density and double-layer, and
12c-formic acid and do not add the light layer of the acid-treated buoyant density of first and carry out PCR-DGGE fingerprint map analyzing (Fig. 5), concrete steps are as follows: use 8% polyacrylamide gel, electrophoretic buffer is 1 × TAE, denatured gradient 45%75%; PCR product applied sample amount is 200 ng DNA; Voltage 80 V, 60 DEG C, electrophoresis 13 h; With SYBR Green I (Invitrogen) (1:10 000, V/V) dyeing 30 min, the rear Gel Doc that uses
tMeQ imager (Bio-Rad) imaging is taken pictures.Result shows
13the ancient flora of product methane of C-formic acid processing buoyant density double-layer falls to forming with other widely different, for example, exist
13the optical density value that C-formic acid is processed band 6,7,8 and 10 in double-layer will obviously be greater than at other buoyant density layer.PCA (PCA) further shows and in different treatment buoyant density layer, produces the fall difference (Fig. 6) of composition of the ancient flora of methane:
13the ancient flora of product methane of C-formic acid processing buoyant density double-layer falls to forming similar, gets together, and falls away from the ancient flora of product methane in the light layer of buoyant density.Finger printing is analyzed consistent declaratives with PCA and has been produced the ancient bacterium metabolism of methane
13c-formic acid, makes self DNA " change is heavy ", thereby appears at buoyant density double-layer, and therefore the ancient flora of the product methane in buoyant density double-layer falls composition with gently layer is obviously different.
(5) species of buoyant density double-layer in finger printing are carried out to phylogenetic tree analysis
To specific band rubber tapping, recovery in PCR-DGGE finger printing, clone, order-checking (Fig. 5), and set up phylogenetic tree analysis (Fig. 7).Result shows
13the ancient bacterium of product methane that C-formic acid is processed in buoyant density double-layer is all under the jurisdiction of
methanobacteriaceae,
methanobacteriaceaeto utilize type to produce the ancient bacterium of methane for main formic acid in examination rice soil.This result has disclosed first formic acid in rice soil and has utilized the ancient bacterium of type product methane, has expanded the cognition of at present being produced from rice field the microbial geochemistry working cycle of the ancient bacterium ecological functions of methane and formic acid driving.
Sequence table
<110> Nanjing Soil Inst., Chinese Academy of Sciences
The DNA stability isotope probe original position of utilizing <120> discloses differentiation rice field formic acid and utilizes type to produce the method for the ancient bacterium of methane
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
ttwagtcagg caacgagc 18
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
tgtgcaagga gcagggac 18
Claims (4)
1. utilize DNA stability isotope probe original position to disclose differentiation rice field formic acid and utilize type to produce the method for the ancient bacterium of methane, it is characterized in that comprising the following steps:
(1) gather rice soil sample;
(2) carry out
13the microcosm of C-formic acid is cultivated experiment;
(3) utilize ultra-high speed whizzer pair
13the microorganism total DNA that C-formic acid is cultivated soil carries out centrifugal layering;
(4) the ancient bacterium gene of the product methane in multiple buoyant densities after layering is carried out to Real-time PCR Analysis and fingerprint map analyzing, judge that the formic acid that whether contains metabolic activity in this rice soil utilizes type to produce the ancient bacterium of methane.
2. utilize according to claim 1 DNA stability isotope probe original position to disclose differentiation rice field formic acid and utilize type to produce the method for the ancient bacterium of methane, the microcosm described in it is characterized in that is cultivated the step of testing and is: by 100 mg
13c-formic acid joins and is equipped with in the 120 mL vials that 5 grams of dry ground are heavy, is adjusted to 60% of the maximum water holding weight of field soil with aseptic deionized water, and by use rubber stopper seal after air in nitrogen replacement vial, to keep the anaerobic environment of culture system; Simultaneously with
12c-formic acid and do not add being treated to of formic acid and be respectively contrast, places 28 DEG C of incubator constant temperature culture, extracts the upper layer of air of 1.2 mL every 3 days out, utilizes gas chromatograph for determination concentration of methane gas to change, and treats that methane concentration starts to reduce to stop cultivating experiment.
3. utilizing according to claim 1 DNA stability isotope probe original position to disclose differentiation rice field formic acid utilizes type to produce the method for the ancient bacterium of methane, it is characterized in that the described centrifugal stratification step of ultra-high speed is: after microcosm cultivation experiment stops, extracting test kit with soil DNA and extract the total DNA of soil microorganisms; Total the soil microorganisms of 2.0 μ g DNA, Gradient Buffer damping fluid and 1.85 g/mL cesium chloride solutions, by 0.1 mL, 0.9 mL and 4.9 mL volume mixture, are got to ultra-high speed whizzer on 5.1 mL mixed solutions; Centrifugal 44 hours of the centrifugal speed of 45 krpm at 20 DEG C; After centrifugal end, the mixed solution in centrifuge tube is carried out to layering with fixed flow rate pump, every layer of 340 μ L volume, points 15 layers; With PEG6000 and every layer of DNA of 70% washing with alcohol.
4. utilizing according to claim 1 DNA stability isotope probe original position to disclose differentiation rice field formic acid utilizes type to produce the method for the ancient bacterium of methane, the determination methods that it is characterized in that described Real-time PCR Analysis and fingerprint map analyzing is: the ancient bacterium Auele Specific Primer of the product methane 1106F/1378R based on as shown in SEQ ID NO.1 and SEQ ID NO.2, utilizes real-time quantitative PCR to analyze the ancient bacterium gene copy number of the product methane in each buoyant density layer; Utilizing PCR-DGGE finger printing and PCA figure to fall to forming to the ancient flora of product methane in buoyant density double-layer and light layer analyzes; Utilize phylogenetic tree analysis to judge and in buoyant density double-layer, produce the ancient fungus kind of methane.
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| CN107942036A (en) * | 2017-11-16 | 2018-04-20 | 中国科学院亚热带农业生态研究所 | A kind of method and device of isotope labeled in situ Methane in Soils oxidation bacteria |
| CN107976348A (en) * | 2017-11-16 | 2018-05-01 | 中国科学院亚热带农业生态研究所 | A kind of method and device of isotope labeled in situ soil methane phase Gu bacterium |
| CN110317863A (en) * | 2019-06-21 | 2019-10-11 | 广东省生态环境技术研究所 | The method of the strain and its key function gene of antimony reduction process is participated in a kind of differentiation soil |
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| CN107942036A (en) * | 2017-11-16 | 2018-04-20 | 中国科学院亚热带农业生态研究所 | A kind of method and device of isotope labeled in situ Methane in Soils oxidation bacteria |
| CN107976348A (en) * | 2017-11-16 | 2018-05-01 | 中国科学院亚热带农业生态研究所 | A kind of method and device of isotope labeled in situ soil methane phase Gu bacterium |
| CN110317863A (en) * | 2019-06-21 | 2019-10-11 | 广东省生态环境技术研究所 | The method of the strain and its key function gene of antimony reduction process is participated in a kind of differentiation soil |
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| CN110317863B (en) * | 2019-06-21 | 2023-07-21 | 广东省科学院生态环境与土壤研究所 | Method for distinguishing strains participating in antimony reduction process in soil and key functional genes thereof |
| CN111485015A (en) * | 2020-04-21 | 2020-08-04 | 浙江工商大学 | Method for separating methane oxidation genome DNA from garbage landfill microorganism |
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| CN112226524A (en) * | 2020-09-09 | 2021-01-15 | 广东省科学院生态环境与土壤研究所 | Method for distinguishing strains participating in nitrate-dependent antimony oxidation process in soil and key functional genes thereof |
| CN112226524B (en) * | 2020-09-09 | 2023-10-27 | 广东省科学院生态环境与土壤研究所 | Method for distinguishing strains participating in nitrate-dependent antimony oxidation process in soil and key functional genes thereof |
| CN113322309A (en) * | 2021-05-13 | 2021-08-31 | 中国科学院南京地理与湖泊研究所 | Method for quantifying microbial environment change response force |
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