CN103966318B - Utilize DNA stability isotope probe original position to disclose and differentiate that rice field formic acid utilizes the method for the ancient bacterium of type methane phase - Google Patents
Utilize DNA stability isotope probe original position to disclose and differentiate that rice field formic acid utilizes the method for the ancient bacterium of type methane phase Download PDFInfo
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Abstract
Utilize DNA stability isotope probe original position to disclose and differentiate that rice field formic acid utilizes the method for the ancient bacterium of type methane phase, comprise the following steps: (1) gathers rice soil sample; (2) carry out
13the microcosm cultural aim of C-formic acid; (3) Ultracentrifuge pair is utilized
13the microorganism total DNA that C-formic acid cultivates soil carries out centrifugal layering; (4) Real-time PCR Analysis and fingerprint map analyzing are carried out to the ancient bacterium gene of the methane phase in buoyant density multiple after layering, judge that the formic acid whether containing metabolic activity in this rice soil utilizes the ancient bacterium of type methane phase.To sum up, does the present invention pass through based on DNA stability isotope probe (DNA-based? Stable? Isotope? Probing) can be sharp, the announcement rice field formic acid of original position utilizes the ancient bacterium of type methane phase, has great meaning for the ecological functions cognition understanding rice field Nutrient Cycling process that microorganism drives and the ancient bacterium functional group of rice field methane phase.
Description
Technical field
The present invention relates to one utilizes DNA stability isotope probe (DNA-basedStableIsotopeProbing) original position announcement differentiation rice field formic acid to utilize the method for the ancient bacterium of type methane phase, belongs to Ecological Distribution of Soil Microorganisms field.
Background technology
Whole world rice field area about 1.5 hundred million hectares, 75% is in waterflooding state.Anaerobic environment makes rice soil contain abundant small molecular organic acid.By these materials of metabolism, microorganism drives 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 up to 150 μMs.In rice soil, multiple-microorganism can metabolism formic acid, as anaerobic bacterium multiple in Clostridia guiding principle can utilize formic acid to perform sulfate reduction and Fe3+ reduction process 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 25-54Tg, wherein formic acid and H
2/ CO
2the methane content produced closes the 10-30% accounting for Estimation.In view of the important prerequisite that formic acid is paddy methane discharge, and the high-content of formic acid in rice soil, we infer that certain in rice soil exist can the methane phase Gu bacterium of direct metabolism formic acid.But formic acid utilizes the ancient bacterium of type methane phase but there is not been reported in rice soil.Its reason the first, current educable microorganism only accounts for 1% of whole microorganism, and a large amount of microorganism can only survive and perform ecological functions in specific habitat, also cannot pure culture; The second, the ancient bacterium of methane phase is strictly anaerobic microorganism, and it is very high to the requirement of growing environment, and traditional method is difficult to screen; Although the 3rd can utilize formic acid to grow the ancient bacterium of pure culture condition lower part methane phase, pure culture condition can not represent the original position habitat in rice soil, so the bacterial strain obtained can not reflect the truth in rice soil.The situ identification that the DNA stability isotope probe technology (DNA-basedStableIsotopeProbing) that developed recently gets up is for we provides possibility.
DNA stability isotope probe combine with technique real-time quantitative PCR and finger printing equimolecular biological method, directedly can excavate the Microbial resources participating in specific ecological process in complicated soil environment, be one of comparatively effective means of current soil functional microorganism situ identification.DNA stability isotope probe technology ultimate principle is the metabolism substrate added in soil containing stable isotope mark, when specific soil microorganisms utilizes this labeled substrate growth and breeding, assimilation substrate will contain the element marked, and for the synthesis of its biomass, as DNA etc.Therefore, by carrying out extraction and isolation qualification and compare of analysis to environment DNA, the functional microorganism driving specific ecological process can be identified in soil.The method accurately can disclose the microorganism performing 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 methane phase, this technology formic acid in rice soil utilizes the application in the ancient dientification of bacteria of type methane phase, will contribute to expanding the cognition of the ecological functions of rice field Nutrient Cycling process that we drive Institute of Micro-biology and the ancient bacterium functional group of rice field methane phase.
Summary of the invention
the technical problem solved:technical problem to be solved by this invention is to provide a kind of DNA stability isotope probe (DNA-basedStableIsotopeProbing) original position to disclose and differentiates that rice field formic acid utilizes the method for the ancient bacterium of type methane phase, to fill up relevant theory blank, the ecological functions cognition for the ancient bacterium functional group of rice field Nutrient Cycling process and rice field methane phase understanding microorganism driving has great contribution.
technical scheme:one of the present invention utilizes DNA stability isotope probe (DNA-basedStableIsotopeProbing) original position announcement differentiation rice field formic acid to utilize the method for the ancient bacterium of type methane phase, and its technical process as shown in Figure 1, specifically comprises the following steps:
(1) rice soil sample is gathered;
(2) carry out
13the microcosm cultural aim of C-formic acid;
(3) Ultracentrifuge pair is utilized
13the soil microorganisms STb gene that C-formic acid is cultivated carries out centrifugal layering;
(4) carry out Real-time PCR Analysis and fingerprint map analyzing to the ancient bacterium gene of the methane phase in buoyant density multiple after layering, the formic acid finally determining to have in this rice soil metabolic activity utilizes the ancient bacterium of type methane phase.
The step of described microcosm cultural aim is: by 100mg
13c-formic acid joins and is equipped with in the heavy 120mL vial of 5 grams of dry ground, is adjusted to 60% of the maximum water holding weight of field soil, and uses rubber stopper seal with after air in nitrogen replacement vial, to keep the anaerobic environment of culture system with aseptic deionized water; Simultaneously with
12c-formic acid and do not add formic acid process for being respectively contrast, place 28 DEG C of incubator constant temperature culture, extracted the upper layer of air of 1.2mL every 3 days out, utilize gas chromatograph for determination concentration of methane gas change, treat that methane concentration starts reduction stopping cultural aim.
Described ultracentrifugation stratification step is: after microcosm cultural aim stops, and extracts test kit extract soil microorganisms STb gene with soil DNA; The soil microorganisms STb gene of 2.0 μ g, GradientBuffer damping fluid and 1.85g/mL cesium chloride solution are pressed 0.1mL, 0.9mL and 4.9mL volume mixture, gets Ultracentrifuge on 5.1mL mixed solution; Centrifugal 44 hours of the centrifugal speed of 45krpm at 20 DEG C; After centrifugal end, with fixed flow rate pump, layering is carried out to the mixed solution in centrifuge tube, every layer of 340 μ L volume, divide 15 layers; With PEG6000 and 70% washing with alcohol every layer of DNA.
The determination methods of described Real-time PCR Analysis and fingerprint map analyzing for: based on the ancient bacterium Auele Specific Primer of the methane phase such as shown in SEQIDNO.1 and SEQIDNO.2 1106F/1378R, utilize real-time quantitative PCR to analyze the ancient bacterium gene copy number of the methane phase in each buoyant density layer; PCR-DGGE finger printing and PCA figure is utilized to fall composition analysis to the ancient flora of the methane phase in buoyant density double-layer and light layer; Phylogenetic tree analysis is utilized to judge the ancient fungus kind of methane phase in buoyant density double-layer.
beneficial effect:to sum up, the present invention by can be sharp based on DNA stability isotope probe (DNA-basedStableIsotopeProbing), the announcement rice field formic acid of original position utilizes the ancient bacterium of type methane phase, has great meaning for the ecological functions cognition understanding rice field Nutrient Cycling process that microorganism drives and the ancient bacterium functional group of rice field methane phase.
Accompanying drawing explanation
Fig. 1 is technological line schematic diagram;
Fig. 2 is discharge of methane change schematic diagram in microcosm cultivating process;
Fig. 3 is methane phase ancient bacterium gene copy number distribution schematic diagram in different buoyant density layer, and wherein A is the ancient bacterium 16SrRNA gene copy number of known methane phase and Cyclethreshold(
c t) typical curve of value and linear equation; B is methane phase ancient bacterium 16SrRNA gene copy number, and PCR primer is through solubility curve (Meltingcurveanalysis) and agarose electrophoresis checking schematic diagram;
Fig. 4 is methane phase ancient bacterium 16SrRNA gene copy number graph of a relation in different buoyant density layer;
Fig. 5 is the finger printing schematic diagram that in different buoyant density layer, the ancient flora of methane phase falls to forming;
Fig. 6 is that in different buoyant density layer, the ancient flora of methane phase falls to forming a point different PCA analysis schematic diagram;
Fig. 7 is that the phylogenetic tree of the ancient bacterium of methane phase analyzes 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 illustration of the present invention instead of restriction the present invention.
embodiment 1
With Jiangdu of Yangzhou City rice soil for research object, application DNA stability isotope probe original position discloses and differentiates that in rice soil, formic acid utilizes the ancient fungus kind of type methane phase.
(1)
13the microcosm cultural aim of C-formic acid
By 100mg's
13c-formic acid joins and is equipped with in the heavy 120mL vial of 5 grams of dry ground, and aseptic deionized water is adjusted to 60% of the maximum water holding weight of field soil, uses rubber stopper seal with after air in nitrogen replacement vial, 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 layer of air on 1.2mL out every 3 days, utilize gas chromatograph for determination concentration of methane gas to change (Fig. 2), treat that methane concentration starts to reduce and stop cultural aim.
(2) ultracentrifugation layering is carried out to soil microorganisms STb gene
After microcosm cultural aim stops, extracting test kit with soil DNA and extract soil microorganisms STb gene; The soil microorganisms STb gene of 2.0 μ g, GradientBuffer damping fluid and 1.85g/mL cesium chloride solution are pressed 0.1mL, 0.9mL and 4.9mL volume mixture, gets Ultracentrifuge on 5.1mL mixed solution; Centrifugal 44 hours of the centrifugal speed (being about 190000 × g) of 45krpm at 20 DEG C; After centrifugal end, with fixed flow rate pump, layering is carried out to the mixed solution in centrifuge tube, every layer of 340 μ L volume, divide 15 layers; With PEG6000 and 70% washing with alcohol every layer of DNA.
(3) analysis of the ancient bacterium gene copy number of the methane phase after layering in each buoyant density
After DNA layering, based on methane phase ancient bacterium Auele Specific Primer 1106F(5'-TTWAGTCAGGCAACGAGC-3')/1378R(5'-TGTGCAAGGAGCAGGGAC-3'), utilize CFX96
tMthermalCycler(Bio-Rad) instrument carries out quantitative PCR mensuration to the ancient bacterium 16SrRNA of the methane phase in each buoyant density layer of different treatment gene fragment, concrete steps are as follows: the typical curve first setting up methane phase ancient bacterium 16SrRNA gene fragment, utilize 1106F/1378R primer pair amplifies methane phase ancient bacterium reference culture 16SrRNA gene fragment, obtain the PCR primer of methane phase ancient bacterium 16SrRNA gene fragment, with Promega Cloning Kit, TA clone is carried out to PCR primer, selected by blue hickie and to test sun with PCR and find clone containing correct clip size, this clone is cultivated expansion in the LB liquid medium containing ammonia benzyl, utilize the plasmid DNA in TakaraMiniBESTPlasmidPurificationKit extraction clone expansion liquid and purifying, ultramicron nucleic acid concentration determinator Nanodrop is utilized to measure plasmid DNA concentration, according to molecular weight and the Avogadro constant (6.02 × 10 of plasmid
23molecule/mol) convert out the copy number containing methane phase ancient bacterium 16SrRNA gene fragment in every microliters plasmid DNA, a series of every microlitre 1.0 × 10 is diluted to 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
tMthermalCycler(Bio-Rad) the ancient bacterium 16SrRNA gene copy number of known methane phase and Cyclethreshold(is set up
c t) typical curve of value and linear equation (Fig. 3 A), real-time fluorescence quantitative PCR system is according to SYBR
premixExTaq tM kit(TaKaRa) test kit explanation.According to this linear equation, utilize external standard method environmentally sample
c tvalue calculates methane phase ancient bacterium 16SrRNA gene copy number in environmental sample, and PCR primer, through solubility curve (Meltingcurveanalysis) and agarose electrophoresis checking (Fig. 3 B), confirms its specificity and validity.
As can be seen from Figure 4
13in the process of C-formic acid, the gene copy number maximum value of the ancient bacterium of methane phase appears at buoyant density double-layer and 1.735g/mL; In contrast,
12in the process of C-formic acid, the maximum gene copy number of the ancient bacterium of methane phase appears at the light layer of buoyant density (1.717g/mL).In addition, add the acid-treated methane phase of first ancient bacterium gene copy number maximum value to be all greater than and not add formic acid process.Have the ancient bacterium of methane phase to utilize formic acid to grow in this result declaratives rice soil, thus its gene copy number increase, in addition
13the ancient bacterium metabolism of methane phase is had in the process of C-formic acid
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 the ancient flora of the methane phase after layering in multiple buoyant density layer falls to forming
Subsequently, right
13the light layer of C-formic acid process 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, and electrophoretic buffer is 1 × TAE, denatured gradient 45%75%; PCR primer applied sample amount is 200ngDNA; Voltage 80V, 60 DEG C, electrophoresis 13h; To dye 30min, rear GelDoc with SYBRGreenI (Invitrogen) (1:10000, V/V)
tMeQimager (Bio-Rad) imaging is taken pictures.Result shows
13the ancient flora of methane phase of C-formic acid process buoyant density double-layer falls to forming with other widely different, such as, exist
13in C-formic acid process double-layer, the optical density value of band 6,7,8 and 10 will obviously be greater than at other buoyant density layer.PCA (PCA) shows the difference (Fig. 6) that in different treatment buoyant density layer, the ancient flora of methane phase falls to forming further:
13the ancient flora of methane phase of C-formic acid process buoyant density double-layer falls to forming similar, gets together, and falls away from the ancient flora of the methane phase in the light layer of buoyant density.Finger printing and PCA analyze the ancient bacterium metabolism of consistent declaratives methane phase
13c-formic acid, make self DNA " become heavy ", thus appear at buoyant density double-layer, the ancient flora of the methane phase therefore in buoyant density double-layer falls to forming obviously different with light layer.
(5) phylogenetic tree analysis is carried out to the species of buoyant density double-layer in finger printing
To the specific band rubber tapping in PCR-DGGE finger printing, reclaim, clone, order-checking (Fig. 5), and set up phylogenetic tree analysis (Fig. 7).Result shows
13ancient bacterium is all under the jurisdiction of methane phase in C-formic acid process buoyant density double-layer
methanobacteriaceae, namely
methanobacteriaceaeutilize the ancient bacterium of type methane phase for main formic acid in examination rice soil.This result discloses formic acid in rice soil first and utilizes the ancient bacterium of type methane phase, has expanded the current cognition to the microbial geochemistry working cycle that the ancient bacterium ecological functions of rice field methane phase and formic acid drive.
Sequence table
<110> Nanjing Soil Inst., Chinese Academy of Sciences
The DNA stability isotope probe original position that utilizes <120> discloses the method that differentiation rice field formic acid utilizes the ancient bacterium of type methane phase
<130>
<160>2
<170>PatentInversion3.3
<210>1
<211>18
<212>DNA
<213> artificial sequence
<400>1
ttwagtcaggcaacgagc18
<210>2
<211>18
<212>DNA
<213> artificial sequence
<400>2
tgtgcaaggagcagggac18
Claims (2)
1. utilize DNA stability isotope probe original position to disclose and differentiate that rice field formic acid utilizes the method for the ancient bacterium of type methane phase, it is characterized in that comprising the following steps:
(1) rice soil sample is gathered;
(2) carry out
13the microcosm cultural aim of C-formic acid: the step of described microcosm cultural aim is: by 100mg
13c-formic acid joins and is equipped with in the heavy 120mL vial of 5 grams of dry ground, is adjusted to 60% of the maximum water holding weight of field soil, and uses rubber stopper seal with after air in nitrogen replacement vial, to keep the anaerobic environment of culture system with aseptic deionized water; Simultaneously with
12c-formic acid and do not add formic acid process for being respectively contrast, place 28 DEG C of incubator constant temperature culture, extracted the upper layer of air of 1.2mL every 3 days out, utilize gas chromatograph for determination concentration of methane gas change, treat that methane concentration starts reduction stopping cultural aim;
(3) Ultracentrifuge pair is utilized
13the microorganism total DNA that C-formic acid cultivates soil carries out centrifugal layering: after microcosm cultural aim stops, and extracts test kit extract soil microorganisms STb gene with soil DNA; The soil microorganisms STb gene of 2.0 μ g, GradientBuffer damping fluid and 1.85g/mL cesium chloride solution are pressed 0.1mL, 0.9mL and 4.9mL volume mixture, gets Ultracentrifuge on 5.1mL mixed solution; Centrifugal 44 hours of the centrifugal speed of 45krpm at 20 DEG C; After centrifugal end, with fixed flow rate pump, layering is carried out to the mixed solution in centrifuge tube, every layer of 340 μ L volume, divide 15 layers; With PEG6000 and 70% washing with alcohol every layer of DNA;
(4) Real-time PCR Analysis and fingerprint map analyzing are carried out to the ancient bacterium gene of the methane phase in buoyant density multiple after layering, judge that the formic acid whether containing metabolic activity in this rice soil utilizes the ancient bacterium of type methane phase.
2. utilize DNA stability isotope probe original position to disclose according to claim 1 and differentiate that rice field formic acid utilizes the method for the ancient bacterium of type methane phase, it is characterized in that the determination methods of described Real-time PCR Analysis and fingerprint map analyzing for: based on the ancient bacterium Auele Specific Primer of the methane phase such as shown in SEQIDNO.1 and SEQIDNO.2 1106F/1378R, utilize real-time quantitative PCR to analyze the ancient bacterium gene copy number of the methane phase in each buoyant density layer; PCR-DGGE finger printing and PCA figure is utilized to fall composition analysis to the ancient flora of the methane phase in buoyant density double-layer and light layer; Phylogenetic tree analysis is utilized to judge the ancient fungus kind of methane phase in buoyant density double-layer.
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| 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 |
| CN107942036B (en) * | 2017-11-16 | 2021-03-12 | 中国科学院亚热带农业生态研究所 | Method and device for in-situ labeling soil methane-oxidizing bacteria by isotope |
| 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 |
| CN111705117A (en) * | 2020-06-10 | 2020-09-25 | 广东工业大学 | Method for in-situ revealing anaerobic iron ammonia oxidizing bacteria in river and lake bottom mud by DNA (deoxyribonucleic acid) stable isotope probe |
| 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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| Title |
|---|
| 稳定性同位素核酸探针技术DNA-SIP 原理与应用;贾仲君;《微生物学报》;20111204;第51卷(第12期);第1585-1594页 * |
| 稻田甲烷排放的微生物群落结构组成及活性初步研究;许静;《中国优秀硕士学位论文全文数据库 农业科技辑》;20130715(第7期);D047-26 * |
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