Method for detecting rhizosphere soil prokaryotic microorganisms of various soybeans based on 16SrDNA deep sequencing

A technology of prokaryotic microorganisms and deep sequencing, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of low detection throughput, small scale, and low coverage of non-culturable microorganisms, and achieve low cost and low cost. Low, reliable results

Inactive Publication Date: 2016-04-27
NANJING UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0006] The problem to be solved in the present invention is to overcome the shortcomings of the prior art, such as low detection throughput, small scale, and low coverage of non-cultivable microorganisms, and provide a method for detecting prokaryotic microorganisms in different soybean rhizosphere soils based on 16SrDNA deep sequencing. Accurate assessment of the effects of genotyped soybeans, including transgenic soybeans and their recipient varieties, on rhizosphere soil prokaryotic microbial communities provides a new approach

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  • Method for detecting rhizosphere soil prokaryotic microorganisms of various soybeans based on 16SrDNA deep sequencing
  • Method for detecting rhizosphere soil prokaryotic microorganisms of various soybeans based on 16SrDNA deep sequencing
  • Method for detecting rhizosphere soil prokaryotic microorganisms of various soybeans based on 16SrDNA deep sequencing

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Embodiment Construction

[0026] 1. Soybean material: Mengdou 12; NZL06-698 contains EPSPS herbicide tolerance gene.

[0027] 2. Field trial design:

[0028] The field test site is in Gongzhuling City, Jilin Province, and each genotype soybean is planted in three or more plots, and the area of ​​each plot is not less than 12m 2 (4m×3m). Each plot has two sampling points, and each sampling point takes two or more plants.

[0029] 3. Sample collection

[0030] First remove ground weeds and litter, remove about 1cm of topsoil on the surface of the field soil, and then take out the soybean plants in the blooming stage (or seedling stage, bulging stage, and mature stage) from the soil and shake it off. Method to take root shaking soil as a system control, mix well and remove root hairs, etc., and store at minus 70°C (at least minus 20°C); then use stroke method to remove the rhizosphere soil that is tightly attached to soybean roots, mix well and remove Root hair, etc., and store at minus 70°C.

[0031] 4. Extract...

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Abstract

The invention belongs to the technical field of soil microbiology, and in particular relates to a method for detecting rhizosphere soil prokaryotic microorganisms of various soybeans based on 16SrDNA deep sequencing. The method comprises the following steps: 1. collecting root shook-off soil and rhizosphere soil of various soybeans in different development stages; 2. extracting microorganism metagenome DNA from the soil; 3. performing PCR amplification on a 16S rDNA fourth hypervariable region in the DNA by virtue of a dual-tag primer so as to construct a library; 4. simultaneously synthesizing and sequencing the qualified library by virtue of a Illumina Miseq platform in a mode of 250 nucleotides at dual ends, so that pure READS is obtained; 5. splicing: clustering at least 38000 effective tags generated from each sample into an operable classifying unit; 6. conducting significance analysis on species composition, structure, diversity and relative abundance difference; and 7. by taking the root shook-off soil as a control group of the system, accurately determining the composition, structure, diversity and relative abundance of a rhizosphere soil prokaryotic microorganism colony, and comparing the various soybeans.

Description

Technical field [0001] The invention belongs to the technical field of soil microbiology, and specifically relates to a method for detecting different soybean rhizosphere soil prokaryotic microorganisms based on 16SrDNA deep sequencing in a field planting mode. Background technique [0002] Rhizosphere is defined as the biologically active area closely surrounding the viable root system, which contains a large number of microorganisms, such as bacteria, actinomycetes and fungi, as well as some algae and viruses. The range is generally within 1 cm from the root surface . In different growth periods of different plants or even the same plant, their root exudates will affect the growth and development of rhizosphere microorganisms; and rhizosphere microorganisms participate in the decomposition of soil organic matter, the formation of humus, the transformation and circulation of nutrients, and many other physiological and biochemical The process, especially the nitrogen-fixing bact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6869
Inventor 杨永华陆桂华戚金亮杨荣武庞延军朱银玲孔令如汤程贻
Owner NANJING UNIV
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