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

A microbial community and plant root technology, applied in the field of indirect extraction of soil DNA, can solve the problems of difficult removal of humus and low DNA recovery rate in the purification step, and achieve the effects of reducing health damage, low cost and strong applicability

Inactive Publication Date: 2012-05-02
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by this invention is to provide a method for indirect extraction of soil DNA for evaluating the diversity of plant root microbial communities, using the average density of bacteria (1.1 μg / cm 3 ) is much smaller than the average density of soil minerals (2.6μg / cm 3 ), the sample is pretreated before cell lysis, and the microbial cells are separated from their soil samples to solve the problem of humus removal difficulties and Problems such as low DNA recovery rate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Add 0.5ml pre-cooled sterile water to 0.5g watermelon root soil sample and mix well, vortex for 10 minutes, then add 0.5ml homogenization buffer A, vortex for 10 minutes, low speed (250g) Centrifuge at room temperature for 10 min, collect the supernatant and transfer to another centrifuge bottle.

[0037] (2) Add 1ml of pre-cooled homogenization buffer B to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, combine the obtained supernatants, and centrifuge at 10000rmp for 30 minutes at room temperature to recover bacteria Somatic cells, discard the supernatant.

[0038](3) Add 1.5 ml of washing solution C, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to precipitate bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K (20mg / ml) to the bacterial cells, bathe in water at 37°C for 30-40min, then add 122ul cell lysate D to the sample, an...

Embodiment 2

[0049] (1) Add 0.5ml pre-cooled sterile water to 0.5g strawberry root soil sample soil sample and mix well, vortex for 10 minutes, then add 0.5ml homogenization buffer A, vortex for 10 minutes, low speed (250g ) at room temperature for 10 min, and the supernatant was collected and transferred to another centrifuge bottle.

[0050] (2) Add 1ml of pre-cooled homogenization buffer B to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, combine the obtained supernatants, and centrifuge at 10000rmp for 30 minutes at room temperature to recover bacteria Somatic cells, discard the supernatant.

[0051] (3) Add 1.5 ml of washing solution C, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to precipitate bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K (20mg / ml) to the bacterial cells, bathe in water at 37°C for 30-40min, then add 122ul cell lysate D t...

Embodiment 3

[0062] (1) Add 0.5ml of pre-cooled sterile water to 0.5g rice root soil sample and mix well, vortex for 10 minutes, then add 0.5ml homogenization buffer A, vortex for 10 minutes, low speed (250g) Centrifuge at room temperature for 10 min, collect the supernatant and transfer to another centrifuge bottle.

[0063] (2) Add 1ml of pre-cooled homogenization buffer B to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, combine the obtained supernatants, and centrifuge at 10000rmp for 30 minutes at room temperature to recover bacteria Somatic cells, discard the supernatant.

[0064] (3) Add 1.5 ml of washing solution C, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to precipitate bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K (20mg / ml) to the bacterial cells, bathe in water at 37°C for 30-40min, then add 122ul cell lysate D to the sample, and ...

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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

technical field [0001] The invention relates to an indirect soil DNA extraction method for evaluating the diversity of plant root microbial communities. Background technique [0002] Most soil microorganisms appear to be extremely adapted to their environment and are not cultureable under normal laboratory conditions. Extracting genomic DNA from the natural environment is a very useful method, which can be used to detect non-culturable microorganisms, track the behavior of some target strains or recombinant genes in the natural environment; it can also be used to reveal the relationship between plant rhizosphere soil microbial ecosystems Genetic diversity and its variation with the environment. This requires the extraction and purification of soil microbial genomic DNA from environmental samples. [0003] The main goal of genomic DNA extraction is to obtain the highest DNA recovery so that the most representative DNA from the microbial community can be obtained for further...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 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
Owner SHANGHAI ACAD OF AGRI SCI
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