CN1740186A

CN1740186A - Fast extraction method of adnascent microbe community total DNA of sponge

Info

Publication number: CN1740186A
Application number: CN 200510029320
Authority: CN
Inventors: 李志勇; 何丽明
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2005-09-01
Filing date: 2005-09-01
Publication date: 2006-03-01
Anticipated expiration: 2025-09-01
Also published as: CN1326866C

Abstract

The fast extraction method of adnascent microbe community total DNA of sponge includes cutting sponge sample into small blocks, suspending on TE buffering liquid and set on ice for direct grinding to cracking sponge tissue, adding lysozyme, sodium dodecyl sulfonate and proteinase K into the cracked sponge, centrifuging and adding Tris-saturated phenol to extract, extraction with Tris-saturated phenol, chloroform and isopentanol solution for several times, adding NaCl and isopropanol to obtain DNA precipitate, washing and dissolving in TE buffering solution, slaking with RNA enzyme, and final agarose gel electrophoresis analysis to detect extracted DNA sample. The said method can obtain total DNA sample of adnascent microbe community in large segment of sponge for the need of adnascent microbe community structure and diversity analysis based on nucleic acid.

Description

The rapid extracting method of adnascent microbe community total DNA of sponge

Technical field

The present invention relates to a kind of rapid extracting method of adnascent microbe community total DNA of sponge, be specifically related to microflora's total DNA extraction method of a kind of suitable sponge symbiotic and epiphyte microorganism population structure composition and diversity analysis.Belong to halobiontic molecular engineering field.

Background technology

Sponge is one of maximum marine organisms of the marine active substance of discovery at present.Assemble a large amount of symbiotic and epiphyte microorganisms in the cavernous body, generally can reach more than 40% of sponge volume.The sponge symbiotic and epiphyte microorganism is considered to the real producer of some sponge active substances.Because the microbe research method that depends on separation and Culture at present only can disclose the microorganism information less than 1%, environmental microorganism more than 99% can't separate at present and obtains, and the research method that therefore depends on separation and Culture can not disclose the composition information of sponge symbiotic and epiphyte microorganism.Molecular engineering based on nucleic acid is formed information and is provided possibility from the relevant sponge functional gene of gene level utilization for we disclose the abundant symbiotic and epiphyte microorganism of sponge.For example: clone library and denaturing gradient gel electrophoresis methods such as (DGGE) based on 16S rDNA have become the strong instrument that discloses complicated microbiota architecture; Functional gene screening, clonal expression approach based on macro genome DNA may for utilizing sponge and symbiotic and epiphyte microorganism functional gene resource thereof to provide.Yet these all are based upon on the high-quality genome DNA extractive technique.Therefore must at first set up efficient and high-quality DNA extraction method.

For sponge symbiotic and epiphyte microorganism total DNA extraction, two lines are arranged generally: the one, carry out DNA extraction behind the first separate microorganism again, another is directly spongy tissue to be carried out cracking to extract DNA.For the former because many microorganisms symbiosiss or colonize in the sponge cell or intercellular mesoplasm layer in, be difficult to make microorganism and sponge cellular segregation.It is also very consuming time promptly to allow to separation, needs specific installation or reagent, easily causes unnecessary degraded.For the latter, that adopts at present directly carries out cracking to spongy tissue and extracts the method for DNA and have following deficiency: generally all need liquid nitrogen, special damping fluid, complicated chemical reagent or dedicated kit, be not suitable for common laboratory and adopt; Simultaneously, extraction step is generally very loaded down with trivial details; The 3rd, these methods are not ideal for the versatility of different sponge sample (often having different spicule structures).

Must embody the microbial diversity information of original position with the nucleic acid samples that micro-flora is formed and diversity is disclosed as purpose, therefore the DNA extraction method that adopts must make the DNA of all microorganisms discharge as far as possible, and avoids unnecessary dna degradation.The skeletal structure of sponge uniqueness has determined the difficulty of DNA extraction.How to extract and high-qualityly can reflect that the DNA sample of original microbial diversity is extremely important, but the easy technology of also not extracting about adnascent microbe community total DNA of sponge at present.Therefore, be necessary very much to develop a kind of suitable different sponge sample fast, be difficult for causing and satisfy the sponge adnascent microbe community DNA extraction method of dna degradation based on the sponge symbiotic and epiphyte microorganism population structure of nucleic acid and the needs of diversity analysis.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, a kind of extracting method quickly and easily that is suitable for the adnascent microbe community total DNA of sponge of different sponge sample is provided, can be easy and extract adnascent microbe community total DNA of sponge efficiently, DNA is fully discharged, avoid degraded, satisfy the needs of diversity analysis.

For realizing this purpose, the present invention is based on the cracking that Mechanical Crushing is finished spongy tissue.Handle through N,O-Diacetylmuramidase, SDS (dodecyl sodium sulfonate is received) and Proteinase K then, adopt the mixing solutions of Tris (Tutofusin tris)-saturated phenol, chloroform, primary isoamyl alcohol and chloroform, primary isoamyl alcohol mixing solutions to extract then successively, add sodium-chlor and Virahol again and obtain the DNA precipitation.Adopt the RNA enzyme to clear up the DNA sample of using the agarose gel electrophoresis analyzing and testing to extract behind the RNA.

Method of the present invention specifically comprises the steps:

1, direct mechanical is ground the cracking spongy tissue: sponge sample is cut into small pieces under aseptic condition, adding is the TE damping fluid of 8.0-8.2 by the pH value of Tutofusin tris and sodium ethylene diamine tetracetate preparation, directly grinds to make the broken cracking of spongy tissue piece become uniform suspension in 5-30 minute on ice cube.

2, cell wall breaking and DNA discharge: get spongy tissue suspension, centrifugal abandon supernatant after, add the TE damping fluid precipitation that suspends, the centrifugal supernatant of abandoning behind the vibration mixing adds the TE damping fluid precipitation that suspends again, and adds behind the N,O-Diacetylmuramidase mixing in 35-40 ℃ of water bath processing.Add 45-50 ℃ of water bath processing behind sodium lauryl sulphate and the Proteinase K mixing again, the centrifuging and taking supernatant adds the abundant mixing of the saturated phenol of isopyknic Tris-and handles, and gets supernatant after centrifugal.

3, adnascent microbe community total DNA of sponge extracts: the saturated phenol of Tris-, chloroform, the primary isoamyl alcohol mixed solution that add 1-1.5 times of volume in above-mentioned supernatant are handled, the saturated phenol of Tris-: chloroform: the volume ratio of primary isoamyl alcohol is 25: 24: 1, behind the centrifuging and taking supernatant, the chloroform, the primary isoamyl alcohol mixed solution that add 1-1.5 times of volume are again handled chloroform: the volume ratio of primary isoamyl alcohol is 24: 1; Behind the centrifuging and taking supernatant, add the 5M NaCl of 0.1-0.2 times of volume and the isopropanol precipitating DNA of 1-1.5 times of volume again; Remove the washing with alcohol precipitation that adds 70-75% behind the Virahol, the centrifugal ethanol that discards, the RNase enzyme that adds TE in the dry postprecipitation and do not contain the DNA enzyme is cleared up RNA 35-40 ℃ of water bath processing; The DNA sample that last agarose gel electrophoresis analyzing and testing is extracted.

Adopt method of the present invention can in 3 hours, obtain the total DNA sample of group of the sponge symbiotic and epiphyte microorganism of big fragment (length reaches more than the 21kb).Be suitable as the high quality template of DGGE (denaturing gradient gel electrophoresis) fingerprinting sponge symbiotic microbial species group structure and multifarious 16S rDNA-PCR amplification, also be suitable as the sample DNA that gene library makes up simultaneously.

Description of drawings

Fig. 1 is a technological line schema of the present invention.

Fig. 2 is the sepharose figure of total DNA of four kinds of sponge symbiotic and epiphyte microorganisms in the embodiment of the invention.

Fig. 3 is grow nonparasitically upon another plant the altogether 16S rDNA amplification of bacterium of four kinds of sponges in the embodiment of the invention.

Embodiment

Below in conjunction with drawings and Examples technical scheme of the present invention is further described.

The technological line of realization the inventive method at first obtains sponge sample as shown in Figure 1, and mechanical mill cracking, spongy tissue also make the cell wall breaking released dna, extracts adnascent microbe community total DNA of sponge.The DNA sample that extracts can be formed and diversity analysis as the microbial population structure.

The present invention carries out in the specific implementation according to the following steps:

1. the mechanical mill cracking of spongy tissue

Get sponge sample 5 grams, be cut into 2mm under the aseptic condition ³Fritter shifts in the mortar be placed in the ice bath, adds the TE damping fluid (pH=8.0) by the EDTA preparation of the Tris of 10mM and 1mM, directly grinds broken cracking spongy tissue, and the time becomes uniform suspension up to the spongy tissue piece about 6-12 minute.

2. cell wall breaking and DNA discharge

(1) suspension of getting 300 μ l spongy tissues is in the centrifuge tube of 1.5ml, and 10, the centrifugal 5min of 000rpm abandons supernatant.

(2) add 1mlTE (pH 8.0) suspension precipitation, behind the 30S that vibrates on the outstanding vibrator in whirlpool, 10, the centrifugal 5min of 000rpm abandons supernatant, and repeats this step once.

(3) add 640 μ lTE (pH 8.0) suspension precipitation, and add 200 μ l N,O-Diacetylmuramidase mixings (10mg/ml), 37 ℃ of water-bath 30min.

(4) add the SDS of 240 μ l 10% and the Proteinase K mixing of 20 μ l 10mg/ml, 55 ℃ of water-bath 30min again.

The centrifugal 10min of (5) 12,000rpm, every pipe get the supernatant of 0.5ml and go in another aseptic 1.5ml centrifuge tube, and add the abundant mixing of the saturated phenol of isopyknic Tris-.

3. the extraction of adnascent microbe community total DNA of sponge

The centrifugal 10min of (1) 13,000rpm gets supernatant and goes in another aseptic 1.5ml centrifuge tube, repeats extracting once with the saturated phenol of Tris-.

(2) supernatant is taken in another aseptic 1.5ml centrifuge tube, adds the saturated phenol of isopyknic Tris-: chloroform: primary isoamyl alcohol (25: 24: 1), mixing.

The centrifugal 5min of (3) 13,000rpm gets supernatant, adds isopyknic chloroform: primary isoamyl alcohol (24: 1) mixing.

The centrifugal 5min of (4) 13,000rpm gets supernatant, adds the 5M NaCl of 0.1 times of volume, adds the Virahol of 1-1.5 times of volume again, and mixing is placed 30min at-20 ℃ of refrigerators gently.

The centrifugal 15min of (5) 14,000rpm discards Virahol, adds the washing with alcohol precipitation of 500 μ l 70%.

The centrifugal 10min of (6) 13,000rpm discards ethanol, and is air-dry, and precipitation adds 30 μ lTE damping fluids, adds the RNase enzyme 5 μ l that do not contain the DNA enzyme, 37 ℃ of water-bath 10min again.

The detection of dyeing of (7) 1.0% agarose gel electrophoresis, EB (ethidium bromide).

Adopt method of the present invention that China's South Sea fine thim asterium sponge, the soft sponge of elephant skin, greedy stubborn sponge and four kinds of sponges of Australian pachydermia sponge have been carried out the extraction of total DNA, obtained high-quality genome DNA sample, the results are shown in Figure 2.A, B, C, D are respectively fine thim asterium sponge, the soft sponge of elephant skin, greedy stubborn sponge and Australian pachydermia sponge among Fig. 2, and ' M ' refers to λ DNA/EcoRI+HindIII ladder, and ' sun ' refers to positive control (the total DNA of E.coli); 6 and 9 be milling time (minute).

Verify total DNA quality of above extraction by the pcr amplification of 16S rDNA.The pcr amplification that the total DNA sample that obtains adopts the TE damping fluid to carry out being directly used in behind the serial dilution 16S rDNA has been obtained good result, and specific implementation method and result are as follows:

With total DNA is template, adopts the 16S rDNA fragment of primer 8f (5 '-GGA GAG TTT GAT CA/CT GGC T-3 ') and 798r (5 '-CCA GGG TAT CTA ATC CTG TT-3 ') amplification sponge DNA of bacteria.

System and reaction conditions are as follows: 9 μ L 10xPCR damping fluid (50mmol/LTris-HCl (pH8.2), 18mmol/L MgCl ₂500mmol/L KCl, 0.1% glycerine, 1% TritonX-100 (Triton X-100)), the primer 8f of 10pmol and the primer 798r of 10pmol, 2 μ L10mmol/L (every kind of base concentration) d NTP, genomic dna that 1 μ L is pure and 2.5U Super Taq enzyme replenish volume to 50 μ L with aseptic deionized water.

The PCR response procedures is as follows: 94 ℃ of pre-sex change 5min, 80 ℃ keep 1min, add Super Taq enzyme.Then by (72 ℃ are extended 2min for 94 ℃ of sex change 1min, 57 ℃ of annealing 1min) 25 circulations, 72 ℃ are extended 2min and finish amplification.Detect the EB observation of dyeing with 1.5% agarose gel electrophoresis.The results are shown in Figure 3.Among Fig. 3, A fine thim asterium sponge, the soft sponge of B elephant skin, the greedy stubborn sponge of C, D Australia pachydermia sponge.As can be seen from Figure 3,16S rDNA band is very pure, and specific amplification is very strong, any assorted band do not occur and pollutes.Illustrate that the sponge microorganism total DNA that extracts with direct polishing has good purity, can be used for molecular biology research.Therefore to be used for the extraction of sponge and the total DNA of symbiotic and epiphyte microorganism thereof be feasible in the present invention.

Claims

1, a kind of rapid extracting method of adnascent microbe community total DNA of sponge is characterized in that comprising the steps:

1) direct mechanical is ground the cracking spongy tissue: sponge sample is cut into small pieces under aseptic condition, adding is the TE damping fluid of 8.0-8.2 by the pH value of Tutofusin tris and sodium ethylene diamine tetracetate preparation, directly grinds to make the broken cracking of spongy tissue piece become uniform suspension in 5-30 minute on ice cube;

2) cell wall breaking and DNA discharge: get spongy tissue suspension, centrifugal abandon supernatant after, add TE damping fluid suspension precipitation, the centrifugal supernatant of abandoning behind the vibration mixing adds the TE damping fluid precipitation that suspends again, and adds behind the N,O-Diacetylmuramidase mixing in 35-40 ℃ of water bath processing, add 45-50 ℃ of water bath processing behind sodium lauryl sulphate and the Proteinase K mixing again, the centrifuging and taking supernatant adds the abundant mixing of the saturated phenol of isopyknic Tris-and handles, and gets supernatant after centrifugal;

3) adnascent microbe community total DNA of sponge extracts: the saturated phenol of Tris-, chloroform, the primary isoamyl alcohol mixed solution that add 1-1.5 times of volume in above-mentioned supernatant are handled, the saturated phenol of Tris-: chloroform: the volume ratio of primary isoamyl alcohol is 25: 24: 1, behind the centrifuging and taking supernatant, the chloroform, the primary isoamyl alcohol mixed solution that add 1-1.5 times of volume are again handled chloroform: the volume ratio of primary isoamyl alcohol is 24: 1; Behind the centrifuging and taking supernatant, add the 5M NaCl of 0.1-0.2 times of volume and the isopropanol precipitating DNA of 1-1.5 times of volume again; Remove the washing with alcohol precipitation that adds 70-75% behind the Virahol, the centrifugal ethanol that discards, the RNase enzyme that adds TE in the dry postprecipitation and do not contain the DNA enzyme is cleared up RNA 35-40 ℃ of water bath processing; The DNA sample that last agarose gel electrophoresis analyzing and testing is extracted.