CN101709298A

CN101709298A - Soil DNA extracting method for evaluating diversity of microbial community of plant root system

Info

Publication number: CN101709298A
Application number: CN200910201072A
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: 2009-12-14
Filing date: 2009-12-14
Publication date: 2010-05-19
Anticipated expiration: 2029-12-14
Also published as: CN101709298B

Abstract

The invention discloses a soil DNA extracting method for evaluating diversity of a microbial community of a plant root system. In the method, pretreatment is carried out on a sample before cell splitting, extracellular DNA and humic substances are removed, and the problems of difficult removal of the humic substances and low DNA recovery rate existing in a purification step are solved. In the extraction process of the method, phenol or chloroform is not used so as to reduce harm on the health of experimenters, obtain complete DNA and molecular fragments greater than 10kb and achieve high yield. OD260/OD230 and OD260/OD280 of the extracted soil microorganism DNA are close to standard values and can be directly applied to molecular operation so as to evaluate the diversity of the microbial community of the plant root system.

Description

A kind of evaluate plant roots that is used for is the multifarious soil DNA extracting method of microflora

Technical field

The present invention relates to a kind of evaluate plant roots that is used for is the multifarious DNA extraction method of soil microbial community, is specifically related to a kind of extracting method that extracts microbe genome DNA from root soil.

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 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 microbe genome DNA extracting method of soil microbial community that technical problem to be solved by this invention provides a kind of evaluate plant roots that is used for, 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:

The described evaluate plant roots that is used for is the multifarious soil DNA extracting method of microflora, may further comprise the steps:

(1) in the 0.1-5g pedotheque, add the 1.5-10mL sample and embathe liquid A, whirlpool concussion 5-20 minute minute, 6000-15000 rev/min, centrifugal 5-10 minute, abandoning supernatant repeated 3 times.

(2) in the soil that obtains by above-mentioned steps (1), add the 1.5mL sample and embathe liquid B, placed on ice 5-30 minute, whirlpool concussion 5-20 minute minute, 6000-15000 rev/min, centrifugal 5-8 minute, abandoning supernatant repeated 3 times.

(3) adding 0.1-1g quartz sand and 1-5 grain diameter in the soil that is obtained by above-mentioned steps (2) is the granulated glass sphere of 4mm, and the phosphoric acid buffer C that adds 978-5000 μ l pH=8.0 is in sample, whirlpool concussion 2-10 minute.

(4) add 100-500 μ l cell pyrolysis liquid D in sample to the soil suspension liquid that obtains by above-mentioned steps (3), whirlpool concussion 5-20 minute, the centrifugal 5-10min of 6000-15000rmp, shard.

(5) will obtain supernatant by above-mentioned steps (4) 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.

(6) will obtain supernatant by above-mentioned steps (5) 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.

(7) will obtain mixed solution by above-mentioned steps (6) 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.

(8) will be placed in the new centrifuge tube by column in above-mentioned steps (7) collection tube, and add 500 μ l washings G in column, the centrifugal 1min of 13000rmp outwells the waste liquid in the collection tube.

(9) will be relay in the recovery collector by column in above-mentioned steps (8) 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.

(10) will relay the recovery collector, the centrifugal 1-5min of 8000-15000rmp by column in above-mentioned steps (9) collection tube.

(11) will be by in the 1.5ml centrifuge tube that column is packed into new in above-mentioned steps (10) 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, in the said extracted method, described sample vat liquor A forms pH=8.0-10 by 20-400mM Tris, 15-200mMEDTA, 50-250mM NaCl, 5wt%TEEPOL, preferably form pH=9.5 by 40mM Tris, 60mM EDTA, 150mMNaCl, 5wt%TEEPOL.

Described vat liquor B is the ethanol of 60-95wt%, preferred 80wt% ethanol.

Described cellular lysate liquid D is made up of 1wt%SDS, 20-60mM Tris, 100-200mM NaCl, 40-100mM EDTA, 0.1wt%PVP, pH=8.0, preferably form pH=8.0 by 1%SDS, 40mM Tris, 150mM NaCl, 60mM EDTA, 0.1%PVP.

Described albumen removal liquid E is made up of 2-5M KAc, 2-8wt% Glacial acetic acid, preferably is made up of 3M KAc, 4% 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 forms 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.

Described elutriant I is 0.1mM Tris-HCl, pH=9.0.

Extracting method of the present invention has the following advantages:

1. DNA 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 extraction method of the present invention is before lysis sample to be carried out pre-treatment, and extracellular dna and soil ulmin are removed, and has avoided purification step to exist soil ulmin to remove difficulty and the low problem of the DNA rate of recovery.

3. DNA extraction method applicability of the present invention is strong, and the OD260/OD230 of the soil microbial DNA of extraction and the OD260/OD280 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 Tris (tricarboxylic ylmethyl aminomethane), EDTA (ethylenediamine tetraacetic acid (EDTA)), NaCl, ethanol, SDS (sodium lauryl sulphate), PVP (polyvinylpyrrolidone), TEEPOL (mixture of Teepol and sodium alkyl benzene sulfonate), KAc (Potassium ethanoate), Glacial acetic acid, guanidinium isothiocyanate, Potassium ethanoate, Trisodium Citrate are homemade analytical pure medicine.

Embodiment 1

(1) to 0.5g west muskmelon root system pedotheque, add the 1.5mL sample and embathe liquid A, whirlpool concussion 10 minutes, 10000 rev/mins, centrifugal 5 minutes, abandoning supernatant repeated 3 times.

(2) add the 1.5mL sample and embathe liquid 2, placed on ice 5 minutes, whirlpool concussion 10 minutes, 10000 rev/mins, centrifugal 5 minutes, abandoning supernatant repeated 3 times.

(3) adding 0.3g quartz sand and 1 diameter is the granulated glass sphere of 4mm, and adds 978 μ l phosphoric acid buffer C (pH=8.0) in sample.Whirlpool concussion 5 minutes.

(4) add 122 μ l cell pyrolysis liquid D in sample, whirlpool concussion 5-20 minute.The centrifugal 10min of 13000rmp, shard.

(5) 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.

(6) 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.

(7) 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.

(8) 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.

(9) 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.

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

In the 1.5ml centrifuge tube of (11) packing into column new, room temperature was placed several minutes, so that the ethanol volatilization is clean, at the careful 30-2 μ l elutriant I that adds 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=2.01, OD ₂₆₀/ OD ₂₈₀=1.86, 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) to 0.5g strawberry root soil sample, add the 1.5mL sample and embathe liquid A, whirlpool concussion 10 minutes minutes, 10000 rev/mins, centrifugal 5 minutes, abandoning supernatant repeated 3 times.

(2) add the 1.5mL sample and embathe liquid 2, placed on ice 5 minutes, whirlpool concussion 10 minutes minutes, 10000 rev/mins, centrifugal 6 minutes, abandoning supernatant repeated 3 times.

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

In the 1.5ml centrifuge tube of (11) packing into column new, room temperature was placed several minutes, 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 1min of 13000rmp is separated DNA in the centrifuge tube, be stored in-20 ℃.

Use spectrophotometric determination OD ₂₃₀, OD ₂₆₀And OD ₂₈₀, the result shows OD ₂₆₀/ OD ₂₃₀=2.04, OD ₂₆₀/ OD ₂₈₀=1.83, 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) to 0.5g rice root pedotheque, add the 1.5mL sample and embathe liquid A, whirlpool concussion 10 minutes minutes, 10000 rev/mins, centrifugal 5 minutes, abandoning supernatant repeated 3 times.

(2) add the 1.5mL sample and embathe liquid 2, placed on ice 5 minutes, whirlpool concussion 10 minutes minutes, 10000 rev/mins, centrifugal 7 minutes, abandoning supernatant repeated 3 times.

(10) 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=1.83, 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 4

(1) to 0.5g Glutinous Semen Maydis root soil sample, add the 1.5mL sample and embathe liquid A, whirlpool concussion 10 minutes minutes, 10000 rev/mins, centrifugal 5 minutes, abandoning supernatant repeated 3 times.

(2) add the 1.5mL sample and embathe liquid 2, placed on ice 5 minutes, whirlpool concussion 10 minutes minutes, 10000 rev/mins, centrifugal 8 minutes, abandoning supernatant repeated 3 times.

(10) 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=2.07, OD ₂₆₀/ OD ₂₈₀=1.82, 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 extracting method of microflora, it is characterized in that, may further comprise the steps:

(1) in the 0.1-5g pedotheque, add the 1.5-10mL sample and embathe liquid A, whirlpool concussion 5-20 minute minute, 6000-15000 rev/min, centrifugal 5-10 minute, abandoning supernatant repeated 3 times;

(2) in the soil that obtains by above-mentioned steps (1), add the 1.5mL sample and embathe liquid B, placed on ice 5-30 minute, whirlpool concussion 5-20 minute minute, 6000-15000 rev/min, centrifugal 5-8 minute, abandoning supernatant repeated 3 times;

(3) adding 0.1-1g quartz sand and 1-5 grain diameter in the soil that is obtained by above-mentioned steps (2) is the granulated glass sphere of 4mm, and the phosphoric acid buffer C that adds 978-5000 μ l pH=8.0 is in sample, whirlpool concussion 2-10 minute;

(4) add 100-500 μ l cell pyrolysis liquid D in sample to the soil suspension liquid that obtains by above-mentioned steps (3), whirlpool concussion 5-20 minute, the centrifugal 5-10min of 6000-15000rmp, shard;

(5) will obtain supernatant by above-mentioned steps (4) 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;

(6) will obtain supernatant by above-mentioned steps (5) 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;

(7) will obtain mixed solution by above-mentioned steps (6) 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;

(8) will be placed in the new centrifuge tube by column in above-mentioned steps (7) collection tube, and add 500 μ l washings G in column, the centrifugal 1min of 13000rmp outwells the waste liquid in the collection tube.

(9) will be relay in the recovery collector by column in above-mentioned steps (8) 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;

(10) will relay the recovery collector, the centrifugal 1-5min of 8000-15000rmp by column in above-mentioned steps (9) collection tube;

(11) will be by in the 1.5ml centrifuge tube that column is packed into new in above-mentioned steps (10) 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 sample vat liquor A forms pH=8.0-10 by 20-400mM Tris, 15-200mM EDTA, 50-250mM NaCl, 5wt%TEEPOL.
3. extracting method according to claim 2 is characterized in that, described sample vat liquor A preferably forms pH=9.5 by 40mM Tris, 60mM EDTA, 150mMNaCl, 5wt%TEEPOL.
4. extracting method according to claim 1 is characterized in that, described sample vat liquor B is the ethanol of 60-95wt%.
5. extracting method according to claim 4 is characterized in that, described vat liquor B is preferably 80wt% ethanol.
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 Tris, 100-200mM NaCl, 40-100mM EDTA, 0.1wt%PVP.
7. extracting method according to claim 6 is characterized in that, described cellular lysate liquid D preferably forms pH=8.0 by 1wt%SDS, 40mM Tris, 150mM NaCl, 60mM EDTA, 0.1wt%PVP.
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.