CN103388030B - Analyzing method for bacterial community structures of rhizosphere soil of peanuts in different growth periods - Google Patents
Analyzing method for bacterial community structures of rhizosphere soil of peanuts in different growth periods Download PDFInfo
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- CN103388030B CN103388030B CN201310351646.XA CN201310351646A CN103388030B CN 103388030 B CN103388030 B CN 103388030B CN 201310351646 A CN201310351646 A CN 201310351646A CN 103388030 B CN103388030 B CN 103388030B
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Abstract
The invention discloses an analyzing method for bacterial community structures of rhizosphere soil of peanuts in different growth periods. The analyzing method comprises the following steps of: collecting the rhizosphere soil of the peanuts in different growth periods and respectively extracting DNA (Deoxyribonucleic Acid) of soil genomes; carrying out amplification by a 16SrRNA (16S ribosomal Ribonucleic Acid) gene degenerate primer to obtain a PCR (Polymerase Chain Reaction) product; carrying out homogenization and then purification on the obtained PCR product; then constructing a 16SrRNA gene library; analyzing and comparing obtained library data so as to compressively display the bacterial community structures of the rhizosphere soil of the peanuts in different growth and development periods. According to the analyzing method disclosed by the invention, all common bacterial species and dominant species of the rhizosphere soil of the peanuts in different growth and development periods can be obtained, and the difference of the bacterial community structures of the rhizosphere soil of the peanuts in different growth and development periods is compressively known. The analyzing method disclosed by the invention has the advantages of high efficiency, short period, and capability of comprehensively displaying the specific bacterial species in the rhizosphere soil.
Description
Technical field
The present invention relates to an analytical procedure of cultivating peanut different growing stage rhizosphere soil bacterial quorum sensing, belong to soil microorganisms and technical field of crop cultivation.
Background technology
Soil microorganisms take part in the working cycle of 80-90% in soil, and soil bacteria is abundance and the highest monoid of diversity in soil microorganisms, in the process such as formation of the degraded of organic matter, the circulation of nutritive element, the accumulation of soil toxic substance and degraded, Soil structure, play very important effect.Rhizosphere (rhizosphere) is the unique astragal formed by the interphase interaction of root system of plant and soil microorganisms, by the impact of root system of plant activity, is all different from root surrounding soil in nature at physics, chemistry and biology.Bacterium in this micro-territory and the interaction between root system of plant more tight, growing of the structure of community of rhizosphere bacteria and plant is closely related.
Peanut is the important oil crops in the world and cash crop, and the feature of its result in underground further increases the uniqueness of its rhizospheric environment.Be limited to current most soil bacteria still not educable present situation, traditional cultural method can not disclose the bacterial structure of peanut rhizosphere soil comprehensively.Due to singularity and the quite much higher sample of soil bacteria of rhizosphere soil sampling, still lack the effective ways of research rhizosphere soil bacterial quorum sensing, the concrete monoid of clear and definite bacterium at present.
Summary of the invention
The object of this invention is to provide the analytical procedure of different growing stage rhizosphere soil bacterial quorum sensing of cultivating peanut, to know that peanut difference is grown the total bacterial groups of rhizosphere soil in period and dominant groups, overall understanding peanut difference is grown the difference of rhizosphere soil bacterial quorum sensing in period.
For achieving the above object, the technology used in the present invention means are: an analytical procedure of cultivating peanut different growing stage rhizosphere soil bacterial quorum sensing, by collecting the rhizosphere soil of peanut different growing stage, extract soil genomic dna respectively, 16S rRNA gene degenerated primer is adopted to increase, the PCR primer obtained is purifying after the process that homogenizes, for building 16S rRNA gene library, the literature data obtained is compared by analysis, thus shows the bacterial quorum sensing of the different rhizosphere soil in period that grows of peanut comprehensively.
As preferably, the rhizosphere soil of described collection peanut different growing stage, its collection method is: irrigated by potted plant peanut with sterilized water, is shoveled out by whole strain peanut plant with scoop, after bulk soil drops, collects the soil being attached to root; Aseptic technique is wanted in whole operating process, and used tool all uses 75% alcohol disinfecting.
As preferably, the rhizosphere soil of described collection peanut different growing stage, select arbitrarily each period three strain peanuts as sample, three soil samples of getting fully mix rear liquid nitrogen flash freezer, frozen for subsequent use in-80 DEG C of Refrigerator stores.
As preferably, described extraction soil genomic dna, whole leaching process must ensure aseptic technique, and all links are all carried out on ice, and agents useful for same also wants 4 DEG C of precoolings.
As preferably, described employing 16S rRNA gene degenerated primer increases, and the primer is:
16S-F:5 '-AGNGTTTGATCMTGGCTCAG-3 ' N:A or G, M:A or C;
16S-R:5 '-GGGCGGWGTGTACAAGKCC-3 ' W:A or T, K:G or C;
Annealing temperature is 55 DEG C, 25 circulations of increasing.
As preferably, described employing degenerated primer increases, institute's genomic dna of carrying is as pcr template, first concentration gradient is set, be respectively 0.1 μ l, 0.25 μ l, 0.5 μ l, 1 μ l and 2 μ l, select optimal concentration after pcr amplification, increase 6 pipes again, often pipe 25 μ l system, after Homogeneous phase mixing, purifying is for building 16S rRNA gene library.
As preferably, all positive colonies in constructed library are checked order by the described literature data com-parison and analysis to obtaining, and adopt the taxon of sequence in each library of DOTUR programanalysis, difference with 3% is standard, and sequence difference≤3% is namely as a taxon.
As preferably, the described literature data com-parison and analysis to obtaining, adopts the log value of OUT frequency, with 2 the end of for, does cluster analysis, to reduce the impact of pcr amplification.
As preferably, the described literature data com-parison and analysis to obtaining take phylogenetic systematics as comparing unit, thus reduces limited the brought sequence alignment error of database registration known array, and the classification of refinement sequence as much as possible.
Beneficial effect of the present invention is: the present invention can know that peanut difference is grown the total bacterial groups of rhizosphere soil in period and dominant groups, overall understanding peanut difference is grown the difference of rhizosphere soil bacterial quorum sensing in period, has that efficiency is high, the cycle is short, an advantage of concrete bacterial groups in comprehensive announcement rhizosphere soil.
Embodiment
Technical scheme of the present invention is further illustrated below by specific embodiment.
One analytical procedure of cultivating peanut different growing stage rhizosphere soil bacterial quorum sensing, take pot experiment, the whole growth cycle of peanut choose seedling stage, the florescence, the berry phase, harvesting time four different times as the time point of research rhizosphere soil bacterial quorum sensing.Equal Stochastic choice three strain in each period peanut, as repeated sample, is collected the rhizosphere soil of three samples respectively, three parts of pedotheques is fully mixed rear liquid nitrogen flash freezer, frozen for subsequent use in-80 DEG C of refrigerators.Sampling process requires aseptic technique, and used tool all uses 75% alcohol disinfecting.Adopt test kit to extract soil genomic dna, whole leaching process ensures aseptic technique, and all links are all carried out on ice, and agents useful for same also wants 4 DEG C of precoolings, to reduce the degradation speed of DNA as far as possible.Choose the suitableeest template concentrations, adopt 16S rRNA gene degenerated primer to increase, the PCR primer of acquisition after the process that homogenizes purifying for building 16S rRNA gene library.
As preferably, the rhizosphere soil of described collection peanut different growing stage, select arbitrarily each period three strain peanuts as sample, three soil samples of getting fully mix rear liquid nitrogen flash freezer, frozen for subsequent use in-80 DEG C of Refrigerator stores.
As preferably, described extraction soil genomic dna, whole leaching process must ensure aseptic technique, and all links are all carried out on ice, and agents useful for same also wants 4 DEG C of precoolings.
As preferably, described employing 16S rRNA gene degenerated primer increases, and the primer is:
16S-F:5 '-AGNGTTTGATCMTGGCTCAG-3 ' N:A or G, M:A or C;
16S-R:5 '-GGGCGGWGTGTACAAGKCC-3 ' W:A or T, K:G or C;
Annealing temperature is 55 DEG C, 25 circulations of increasing.
As preferably, described employing degenerated primer increases, institute's genomic dna of carrying is as pcr template, first concentration gradient is set, be respectively 0.1 μ l, 0. 25 μ l, 0. 5 μ l l, 1 μ l and 25 μ l, select optimal concentration after pcr amplification, increase 6 pipes again, often pipe 25 μ l system, after Homogeneous phase mixing, purifying is for building 16S rRNA gene library.
As preferably, all positive colonies in constructed library are checked order by the described literature data com-parison and analysis to obtaining, and adopt the taxon of sequence in each library of DOTUR programanalysis, difference with 3% is standard, and sequence difference≤3% is namely as a taxon.
As preferably, the described literature data com-parison and analysis to obtaining, adopts the log value of OUT frequency, with 2 the end of for, does cluster analysis, to reduce the impact of pcr amplification.
As preferably, the described literature data com-parison and analysis to obtaining take phylogenetic systematics as comparing unit, thus reduces limited the brought sequence alignment error of database registration known array, and the classification of refinement sequence as much as possible.
The literature data of gained is carried out com-parison and analysis.Four constructed 16S rRNA gene libraries, altogether acquisition 415 positive colonies, identify 253 OTUs(wherein 7 be mosaic).Cluster analysis shows, the rhizosphere soil bacterial structure difference in four periods is little, and the berry phase is more similar with the bacterial structure of harvesting time.Through compare of analysis, identify altogether 21 monoids relevant to bacterium, wherein acidfast bacilli door, actinomycetes door, Firmicutes, green curved bacterium door, bud Zymomonas mobilis door, floating mustiness bacterium door, protein fungus door and wart germ door are dominant groups in all libraries.Specific to order, acidfast bacilli order, painted Zoopagales, bud unit cell Zoopagales, myxococcus order and sphingolipid unit cell Zoopagales, actinomycetales, Burkholderia order and Pseudomonadales are the dominant groups had four periods of growing.Diversity and the abundance of Burkholderia order and wherein a unknown γ-albumen Gammaproteobacteria bacterium show trend of obvious reduced in the whole period of growing, and Bdellovibrio order, warm rope Pseudomonas in green curved Zoopagales,
solirubrobacteralesobvious increase trend is then presented with the diversity of a unknown alpha 2 delta-protein Gammaproteobacteria bacterium and abundance.
The present invention includes but be not limited to following embodiment, according to the change not departing from flesh and blood of the present invention that prior art is carried out the present invention, still belonging to protection scope of the present invention.
Claims (2)
1. an analytical procedure of cultivating peanut different growing stage rhizosphere soil bacterial quorum sensing, it is characterized in that: by collecting the rhizosphere soil of peanut different growing stage, extract soil genomic dna respectively, 16S rRNA gene degenerated primer is adopted to increase, the PCR primer obtained is purifying after the process that homogenizes, for building 16S rRNA gene library, the literature data obtained is compared by analysis, thus show the bacterial quorum sensing of the different rhizosphere soil in period that grows of peanut comprehensively;
Described employing 16S rRNA gene degenerated primer increases, and the primer is:
16S-F:5 '-AGNGTTTGATCMTGGCTCAG-3 ' N: A or G, M: A or C;
16S-R:5 '-GGGCGGWGTGTACAAGKCC-3 ' W: A or T, K: G or C;
Annealing temperature is 55 DEG C, 25 circulations of increasing;
Described employing degenerated primer increases, institute's genomic dna of carrying is as pcr template, first concentration gradient is set, be respectively 0.1 μ l, 0.25 μ l, 0.5 μ l, 1 μ l and 2 μ l, optimal concentration is selected after PCR amplification, again increase 6 pipes, often pipe 25 μ l system, and after Homogeneous phase mixing, purifying is for building 16S rRNA gene library;
The described literature data com-parison and analysis to obtaining, adopts the log value of OUT frequency, with 2 the end of for, does cluster analysis, to reduce the impact of PCR amplification.
2. according to the analytical procedure of the peanut different growing stage rhizosphere soil bacterial quorum sensing described in claim 1, it is characterized in that: the rhizosphere soil of described collection peanut different growing stage, its collection method is: irrigated by potted plant peanut with sterilized water, with scoop, whole strain peanut plant is shoveled out, after bulk soil drops, collect the soil being attached to root; Aseptic technique is wanted in whole operating process, and used tool all uses 75% alcohol disinfecting.
3. according to the analytical procedure of the peanut different growing stage rhizosphere soil bacterial quorum sensing described in claim 1, it is characterized in that: the rhizosphere soil of described collection peanut different growing stage, select arbitrarily each period three strain peanuts as sample, three soil samples of getting fully mix rear liquid nitrogen flash freezer, frozen for subsequent use in-80 DEG C of Refrigerator stores.
4. according to the analytical procedure of the peanut different growing stage rhizosphere soil bacterial quorum sensing described in claim 1, it is characterized in that: described extraction soil genomic dna, whole leaching process must ensure aseptic technique, and all links are all carried out on ice, and agents useful for same also wants 4 DEG C of precoolings.
5. according to the analytical procedure of the peanut different growing stage rhizosphere soil bacterial quorum sensing described in claim 1, it is characterized in that: the described literature data com-parison and analysis to obtaining, all positive colonies in constructed library are checked order, adopt the taxon of sequence in each library of DOTUR programanalysis, difference with 3% is standard, and sequence difference≤3% is namely as a taxon.
6. according to the analytical procedure of the peanut different growing stage rhizosphere soil bacterial quorum sensing described in claim 1, it is characterized in that: the described literature data com-parison and analysis to obtaining, take phylogenetic systematics as comparing unit, thus reduce limited the brought sequence alignment error of database registration known array, and the classification of refinement sequence as much as possible.
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Non-Patent Citations (2)
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| Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA;JULIAN R. MARCHESI;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19981231;第64卷(第2期);第797页 * |
| 王小兵.红壤连作花生不同生育期根际微生物区系变化研究.《扬州大学学报(农业与生命科学版)》.2011,第32卷(第4期), * |
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