CN101717771A

CN101717771A - Soil DNA indirect extraction method for evaluating diversity of plant root system microflora

Info

Publication number: CN101717771A
Application number: CN200910200685A
Authority: CN
Inventors: 唐雪明; 王金斌; 赵凯; 谭芙蓉; 吴潇; 朱宏; 陶世如; 蒋玲曦; 王利刚; 刘华
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2009-12-24
Filing date: 2009-12-24
Publication date: 2010-06-02
Anticipated expiration: 2029-12-24
Also published as: CN101717771B

Abstract

The invention discloses a soil DNA indirect extraction method for evaluating the diversity of a plant root system microflora. Because the mean density of bacteria is far smaller than the mean density of soil minerals, a sample is pretreated before cell lysis, and microbial cells are separated from the soil sample, thereby avoiding the problems of difficult humus removal and low DNA recovery rate occurring in the purification process. In the extraction process, the invention does not use phenols or chloroform, the damage to the health of an experimenter is reduced, the obtained DNA is complete, a molecular segment is larger than 10kb, and the yield is high. The OD260/OD230 and the OD260/OD280 of the extracted soil microbe DNA approach to a standard value, and the soil DNA indirect extraction method can be directly applied to molecular operation so as to evaluate the diversity of the plant root system microflora.

Description

Being used for evaluate plant roots is the multifarious soil DNA indirect extraction method of microflora

Technical field

The present invention relates to a kind of evaluate plant roots that is used for is the multifarious soil DNA indirect extraction method of microflora.

Background technology

As if the most of soils microorganism extremely adapt to its residing environment and can not cultivate under general laboratory condition.Extract genomic dna and be very useful method from physical environment, can be used for detecting can not cultured microorganism, follows the tracks of the behavior in physical environment of some aimed strain or recombination; Also can be used for disclosing the diversity of the gene in the plant rhizosphere soil microbial ecosystem and with the variation of environment.This just requires extracting and purifying soil microbe genome DNA from environmental sample.

The major objective of extracting genome DNA is to obtain the highest DNA to reclaim, thereby obtains the most representative DNA from microflora, and carries out further molecule manipulation (as PCR, SSCP, DGGE, TGGE, AFLP etc.).But can not get rid of in leaching process from the detritus acid that the sample in the environment contains in very complicated composition, the especially soil, soil ulmin has the physicochemical property that are similar to nucleic acid.Therefore, soil ulmin is extracted with DNA jointly together with the organic molecule that is adsorbed.Humic acid directly influences subsequent P CR amplification, hybridization, restriction endonuclease digestion and bacterium conversion etc.In most of grand genome research, isolate the macro genome DNA that does not contain soil ulmin, remain a difficult problem.In addition, slightly carry each purification step of DNA after the lysis, for example the repeated purification step that carried out before dna molecular research has inevitably caused the loss of DNA.All there is defective in most gene group DNA indirect extraction method, and for example lysis is incomplete, and DNA is adsorbed in upper soll layer, contains losing, degrade and shearing of enzyme inhibitors and DNA in the soil extract thing.

Therefore, need research be applicable to convenient, fast, the practical method that the plant rhizosphere soil microbial DNA extracts, use DNA sample that ordinary method was obtained to have PCR inhibition such as humic acid and problem such as the lysis rate is low, the DNA loss is serious to solve.

Summary of the invention

It is the multifarious soil DNA indirect extraction method of microflora that technical problem to be solved by this invention is to provide a kind of evaluate plant roots that is used for, and utilizes mean density (the 1.1 μ g/cm of bacterium ³) much smaller than mean density (the 2.6 μ g/cm of soil mineral ³), before lysis, sample is carried out pre-treatment, microorganism cells is separated from their pedotheques, after being soil microorganisms lysis with solution, because soil ulmin has the physicochemical property that are similar to nucleic acid, purification step exists soil ulmin to remove problems such as difficulty and the DNA rate of recovery are low.

In order to achieve the above object, the present invention realizes by the following technical solutions:

A kind of evaluate plant roots that is used for is the multifarious soil DNA indirect extraction method of microflora, may further comprise the steps:

(1) in the 0.1-5g pedotheque, add the sterilized water mixing of 0.1-5mL precooling after, whirlpool concussion 5-20 minute minute adds 0.1-5mL homogenate buffer A then, whirlpool concussion 5-20 minute, the centrifugal 10min of low speed (50-400g) room temperature collects supernatant liquor and is transferred in another centrifugal bottle.

(2) the homogenate buffer B washing of adding 0.2-10mL precooling is resuspended in the throw out that is obtained by above-mentioned steps (1), whirlpool concussion 5-20 minute, and the centrifugal 10min of low speed (50-400g) room temperature gets supernatant liquor, merges with step (1) gained supernatant liquor.

(3) supernatant liquor that will obtain by above-mentioned steps (2), under the room temperature the centrifugal 10-60 of 6000-15000rmp minute to reclaim somatic cells.

(4) the washings C of adding 1-150ml in the somatic cells that is obtained by above-mentioned steps (3) washed 2-10 minute, and wherein washings C was the 1g/L trisodium phosphate with the precipitation somatic cells in the centrifugal 10-60 of 6000-15000rmp minute under the room temperature.

(5) add 160ul N,O-Diacetylmuramidase (50mg/ml) and 20ul Proteinase K (20mg/ml) to the somatic cells that obtains by above-mentioned steps (4), 37 ℃ of water-bath 10-40min, add 100-500 μ l cell pyrolysis liquid D then in sample, put upside down gently mixing 1-10 minute, the centrifugal 5-10min of 6000-15000rmp, shard.

(6) will obtain supernatant by above-mentioned steps (5) transfers in the clean centrifuge tube, add 250-1500 μ l albumen and remove liquid E in pipe, gently put upside down mixing 10 times with have gentle hands, room temperature was placed 5-10 minute, and the centrifugal 3-10min of 8000-15000rmp is with precipitating proteins.

(7) will obtain supernatant by above-mentioned steps (6) and transfer in the clean centrifuge tube, and add 3 times of volume DNA and connect liquid F in centrifuge tube, put upside down mixing 1-5min with hand.

(8) will obtain mixed solution by above-mentioned steps (7) adds in the 2ml collection tube in the column, room temperature is placed 1-5min, the centrifugal 1-3min of 3000-10000rmp, outwell the waste liquid in the collection tube, column is reentered in the 2ml centrifuge tube, it is centrifugal to add supernatant liquor once more, finishes until all supernatant liquors are centrifugal.

(9) will be placed in the new centrifuge tube by column in above-mentioned steps (8) collection tube, and add 500 μ l washings G in column, the centrifugal 1min of 13000rmp outwells the waste liquid in the collection tube.

(10) will be relay in the recovery collector by column in above-mentioned steps (9) collection tube, and add 650 μ l washings H in column, centrifugal 1 minute of 8000-15000rpm outwells waste liquid in the collection tube, repeats above-mentioned steps once.

(11) will relay the recovery collector, the centrifugal 1-5min of 8000-15000rmp by column in above-mentioned steps (10) collection tube.

(12) will be by in the 1.5ml centrifuge tube that column is packed into new in above-mentioned steps (11) collection tube, room temperature is placed, so that the ethanol volatilization is clean, at the careful 30-50 μ l elutriant I that adds of film central authorities, do not poke film, room temperature is placed 2min, the centrifugal 1-3min of 8000-15000rmp, be separated DNA in the centrifuge tube, be stored in-20 ℃.Wherein elutriant I is 0.1mM Tris-HCl, pH=9.0.

Wherein, in the said extracted method, described homogenate buffer A forms pH=6.0-9.0 by 20-400mM Tris, 15-200mMEDTA, 50-250mM NaCl, 0.5-3%pvpp; Preferably form pH=7.5 by 200mM Tris, 200mM EDTA, 300mMNaCl, 2%pvpp.

Described homogenate buffer B is that 10-200mM Tris, 5-100mM EDTA, 25-150mM NaCl, 0.2-1.5%pvpp form pH=6.0-9.0; Preferably form pH=7.5 by 100mM Tris, 100mM EDTA, 150mMNaCl, 1%pvpp.

Described cell pyrolysis liquid D forms pH=8.0 by 1wt%SDS, 20-60mM Tris, 100-200mM NaCl, 40-100mM EDTA; Preferably form pH=8.0 by 1%SDS, 40mM Tris, 150mM NaCl, 60mM EDTA.

Described albumen is removed liquid E and is made up of 2-5M KAc, 2-8wt% Glacial acetic acid; Preferably form by 3M KAc, 4wt% Glacial acetic acid.

Described DNA connects liquid F and is made up of 6-10M guanidinium isothiocyanate, 200-600mM Potassium ethanoate, and pH is 4.5-6.0; Preferably form pH=5.5 by 8M guanidinium isothiocyanate, 500mM Potassium ethanoate.

Described washings G is made up of 5.5M guanidinium isothiocyanate, 23mM Trisodium Citrate.

Described washings H forms pH=5.0 by 70wt% ethanol, 200mM Potassium ethanoate.

Indirect extraction method of the present invention has the following advantages:

1. DNA indirect extraction method of the present invention is simple to operate, does not need comparatively complex apparatus, and conventional biological chemistry or microbiology laboratory just can be finished; And the reagent that relates to is the reagent of routine analysis, molecular biology use, and is low with respect to the test kit cost on the market.

2. DNA indirect extraction method of the present invention is before lysis sample to be carried out pre-treatment, and microorganism cells is separated from their pedotheques, has avoided purification step to exist soil ulmin to remove difficulty and the low problem of the DNA rate of recovery.

3. DNA indirect extraction method suitability of the present invention is strong, the OD of the soil microbial DNA of extraction ₂₆₀/ OD ₂₃₀And OD ₂₆₀/ OD ₂₈₀The value of being near the mark can directly apply to molecule manipulation, and coming evaluate plant roots is microflora's diversity.

4. leaching process does not use phenol, chloroform, and the DNA that obtains is complete to reduce healthy injury, institute to the experimenter, and molecule fragment is greater than 10kb, the output height.

Embodiment

Below in conjunction with specific embodiment technical scheme of the present invention is described in further detail.

Reagent such as tricarboxylic ylmethyl aminomethane (Tris), disodium ethylene diamine tetraacetate (EDTA), NaCl, ethanol, sodium lauryl sulphate (SDS), pvpp (cross-linking polyethylene pyrrolidone, the g/L of percentage composition unit), trisodium phosphate, Potassium ethanoate (KAc), Glacial acetic acid, guanidinium isothiocyanate, Trisodium Citrate are homemade analytical pure medicine.

N,O-Diacetylmuramidase and Proteinase K are that worker's import packing is given birth in Shanghai.

Embodiment 1

(1) in the muskmelon root system pedotheque of 0.5g west, add the sterilized water mixing of 0.5ml precooling after, whirlpool concussion 10 minutes minutes adds 0.5ml homogenate buffer A then, whirlpool concussion 10 minutes, the centrifugal 10min of low speed (250g) room temperature collects supernatant liquor and is transferred in another centrifugal bottle.

(2) the homogenate buffer B washing of adding 1ml precooling is resuspended, whirlpool concussion 10 minutes, and the centrifugal 10min of low speed (250g) room temperature gets supernatant liquor, merges the gained supernatant liquor, and 10000rmp abandoned supernatant to reclaim somatic cells in centrifugal 30 minutes under the room temperature.

(3) add the washings C of 1.5ml, wash 5 minutes, centrifugal 30 minutes of 10000rmp is to precipitate somatic cells under the room temperature.Add 160ul N,O-Diacetylmuramidase (50mg/ml) and 20ul Proteinase K (20mg/ml) to somatic cells, 37 ℃ of water-bath 30-40min add 122 μ l cell pyrolysis liquid D then in sample, whirlpool concussion 5-20 minute.The centrifugal 10min of 13000rmp, shard.

(4) supernatant is transferred in the clean centrifuge tube, added 250 μ l albumen and remove liquid E in pipe, gently put upside down mixing 10 times with have gentle hands, room temperature was placed 5 minutes, the centrifugal 5min of 13000rmp.

(5) supernatant is transferred in the clean centrifuge tube, added 3 times of volume DNA and connect liquid F in centrifuge tube, put upside down mixing 1-5min with hand.

(6) mixed solution is added in the 2ml collection tube in the column, room temperature is placed 2min.The centrifugal 1min of 6000rmp outwells the waste liquid in the collection tube, and column is reentered in the 2ml centrifuge tube, and it is centrifugal to add supernatant liquor once more, finishes until all supernatant liquors are centrifugal.

(7) column is placed in the new centrifuge tube, adds 500 μ l washings G in column, the centrifugal 1min of 13000rmp outwells the waste liquid in the collection tube.

(8) column is relay in the recovery collector, and add 650 μ l washings H in column, centrifugal 1 minute of 13000rpm outwells waste liquid in the collection tube, repeats above-mentioned steps once.

(9) column is relay the recovery collector, the centrifugal 2min of 13000rmp.

In the 1.5ml centrifuge tube of (10) packing into column new, room temperature was placed several minutes, so that the ethanol volatilization is clean, at the careful 30 μ l elutriant I that add of film central authorities.Do not poke film, room temperature is placed 2min.The centrifugal 1min of 13000rmp is separated DNA in the centrifuge tube, be stored in-20 ℃.

The add-on of above solution all can be according to the increase of the amount of extracting sample, and proportional increase.

Use spectrophotometric determination OD ₂₃₀, OD ₂₆₀And OD ₂₈₀, the result shows: OD ₂₆₀/ OD ₂₃₀=2.00, OD ₂₆₀/ OD ₂₈₀=1.81, approach standard value and (annotate: OD ₂₆₀/ OD ₂₃₀Standard value is 2.0, OD ₂₆₀/ OD ₂₈₀Standard value is 1.6), can directly apply to molecule manipulation, estimating evaluate plant roots is microflora's diversity.

Embodiment 2

(1) in 0.5g strawberry root soil sample pedotheque, add the sterilized water mixing of 0.5ml precooling after, whirlpool concussion 10 minutes minutes adds 0.5ml homogenate buffer A then, whirlpool concussion 10 minutes, the centrifugal 10min of low speed (250g) room temperature collects supernatant liquor and is transferred in another centrifugal bottle.

(9) column is relay the recovery collector, the centrifugal 2min of 13000rmp.

Use spectrophotometric determination OD ₂₃₀, OD ₂₆₀And OD ₂₈₀, the result shows OD ₂₆₀/ OD ₂₃₀=2.01, OD ₂₆₀/ OD ₂₈₀=1.81, approach standard value and (annotate: OD ₂₆₀/ OD ₂₃₀Standard value is 2.0, OD ₂₆₀/ OD ₂₈₀Standard value is 1.6), can directly apply to molecule manipulation, estimating evaluate plant roots is microflora's diversity.

Embodiment 3

(1) on the 0.5g rice root, add the sterilized water mixing of 0.5ml precooling in the earth sample after, whirlpool concussion 10 minutes minutes adds 0.5ml homogenate buffer A then, whirlpool concussion 10 minutes, the centrifugal 10min of low speed (250g) room temperature collects supernatant liquor and is transferred in another centrifugal bottle.

(9) column is relay the recovery collector, the centrifugal 2min of 13000rmp.

Embodiment 4

(1) in 0.5g Glutinous Semen Maydis root soil sample, add the sterilized water mixing of 0.5ml precooling after, whirlpool concussion 10 minutes minutes adds 0.5ml homogenate buffer A then, whirlpool concussion 10 minutes, the centrifugal 10min of low speed (250g) room temperature collects supernatant liquor and is transferred in another centrifugal bottle.

(9) column is relay the recovery collector, the centrifugal 2min of 13000rmp.

Use spectrophotometric determination OD ₂₃₀, OD ₂₆₀And OD ₂₈₀, the result shows OD ₂₆₀/ OD ₂₃₀=2.02, OD ₂₆₀/ OD ₂₈₀=1.81, approach standard value and (annotate: OD ₂₆₀/ OD ₂₃₀Standard value is 2.0, OD ₂₆₀/ OD ₂₈₀Standard value is 1.6), can directly apply to molecule manipulation, estimating evaluate plant roots is microflora's diversity.

Should be noted that at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.

Claims

One kind to be used for evaluate plant roots be the multifarious soil DNA indirect extraction method of microflora, it is characterized in that, may further comprise the steps:

(1) in the 0.1-5g pedotheque, add the sterilized water mixing of 0.1-5ml precooling after, whirlpool concussion 5-20 minute minute adds 0.1-5ml homogenate buffer A then, whirlpool concussion 5-20 minute, the centrifugal 10min of low speed 50-400g room temperature collects supernatant liquor and is transferred in another centrifugal bottle;

(2) the homogenate buffer B washing of adding 0.2-10mL precooling is resuspended in the throw out that is obtained by above-mentioned steps (1), whirlpool concussion 5-20 minute, and the centrifugal 10min of low speed 50-400g room temperature gets supernatant liquor, merges with step (1) gained supernatant liquor;

(3) supernatant liquor that will obtain by above-mentioned steps (2), under the room temperature the centrifugal 10-60 of 6000-15000rmp minute to reclaim somatic cells;

(4) the washings C of 1-150ml in the somatic cells that is obtained by above-mentioned steps (3) washed 2-10 minute, and wherein washings C was the 1g/L trisodium phosphate with the precipitation somatic cells in the centrifugal 10-60 of 6000-15000rmp minute under the room temperature;

(5) add 160ul N,O-Diacetylmuramidase and 20ul Proteinase K to the somatic cells that obtains by above-mentioned steps (4), 37 ℃ of water-bath 10-40min add 100-500 μ l cell pyrolysis liquid D then in sample, put upside down gently mixing 1-10 minute, the centrifugal 5-10min of 6000-15000rmp, shard;

(6) will obtain supernatant by above-mentioned steps (5) transfers in the clean centrifuge tube, add 250-1500 μ l albumen and remove liquid E in pipe, gently put upside down mixing 10 times with have gentle hands, room temperature was placed 5-10 minute, and the centrifugal 3-10min of 8000-15000rmp is with precipitating proteins;

(7) will obtain supernatant by above-mentioned steps (6) and transfer in the clean centrifuge tube, and add 3 times of volume DNA and connect liquid F in centrifuge tube, put upside down mixing 1-5min with hand;

(8) will obtain mixed solution by above-mentioned steps (7) adds in the 2ml collection tube in the column, room temperature is placed 1-5min, the centrifugal 1-3min of 3000-10000rmp, outwell the waste liquid in the collection tube, column is reentered in the 2ml centrifuge tube, it is centrifugal to add supernatant liquor once more, finishes until all supernatant liquors are centrifugal;

(9) will be placed in the new centrifuge tube by column in above-mentioned steps (8) collection tube, and add 500 μ l washings G in column, the centrifugal 1min of 13000rmp outwells the waste liquid in the collection tube.

(10) will be relay in the recovery collector by column in above-mentioned steps (9) collection tube, and add 650 μ l washings H in column, centrifugal 1 minute of 8000-15000rpm outwells waste liquid in the collection tube, repeats above-mentioned steps once;

(11) will relay the recovery collector, the centrifugal 1-5min of 8000-15000rmp by column in above-mentioned steps (10) collection tube;

(12) will be by in the 1.5ml centrifuge tube that column is packed into new in above-mentioned steps (11) collection tube, room temperature is placed, so that the ethanol volatilization is clean, at the careful 30-50 μ l elutriant I that adds of film central authorities, do not poke film, room temperature is placed 2min, the centrifugal 1-3min of 8000-15000rmp, be separated DNA in the centrifuge tube, be stored in-20 ℃.Wherein elutriant I is 0.1mM Tris-HCl, pH=9.0.
2. extracting method according to claim 1 is characterized in that, described homogenate buffer A forms pH=6.0-9.0 by 20-400mM Tris, 15-200mM EDTA, 50-250mM NaCl, 0.5-3%pvpp.
3. extracting method according to claim 2 is characterized in that, described homogenate buffer A preferably forms pH=7.5 by 200mM Tris, 200mM EDTA, 300mMNaCl, 2%pvpp.
4. extracting method according to claim 1 is characterized in that, described homogenate buffer B forms pH=6.0-9.0 by 10-200mM Tris, 5-100mM EDTA, 25-150mM NaCl, 0.2-1.5%pvpp.。
5. extracting method according to claim 4 is characterized in that, described homogenate buffer B preferably forms pH=7.5 by 100mM Tris, 100mM EDTA, 150mMNaCl, 1%pvpp.
6. extracting method according to claim 1 is characterized in that, described cell pyrolysis liquid D forms pH=8.0 by 1wt%SDS, 20-60mM Tri s, 100-200mM NaCl, 40-100mM EDTA.
7. extracting method according to claim 6 is characterized in that, described cellular lysate liquid D preferably forms pH=8.0 by 1%SDS, 40mM Tris, 150mM NaCl, 60mM EDTA.
8. extracting method according to claim 1 is characterized in that, described albumen is removed liquid E and is made up of 2-5M KAc, 2-8wt% Glacial acetic acid.
9. extracting method according to claim 8 is characterized in that, described albumen is removed liquid E and preferably is made up of 3M KAc, 4wt% Glacial acetic acid.
10. extracting method according to claim 1 is characterized in that, described DNA connects liquid F and is made up of 6-10M guanidinium isothiocyanate, 200-600mM Potassium ethanoate, and pH is 4.5-6.0.
11. extracting method according to claim 10 is characterized in that, described DNA connects liquid F and preferably forms pH=5.5 by 8M guanidinium isothiocyanate, 500mM Potassium ethanoate.
12. extracting method according to claim 1 is characterized in that, described washings G is made up of 5.5M guanidinium isothiocyanate, 23mM Trisodium Citrate.
13. extracting method according to claim 1 is characterized in that, described washings H forms pH=5.0 by 70wt% ethanol, 200mM Potassium ethanoate.
14. extracting method according to claim 1 is characterized in that, described lysozyme concentration is 50mg/mL, and the concentration of described Proteinase K is 20mg/mL.