High-throughput separation culture method for crop root system microbiome

Abstract

The invention discloses a high-throughput separation culture method for crop root system microbiome. The method comprises the following steps of S1, performing gradient dilution separation culture oncrop root system bacteria; S2, extracting DNA of cultured bacteria; S3, carrying out two-step PCR on DNA sub-holes, namely amplifying 16S variable regions V5-V7 by taking 799F and 1193R as primers inthe first step and taking 799F-2 containing hole barcode and 1193R-2 containing plate barcode as primers in the second step to obtain a bacterium identification library; S4, carrying out sequencing analysis; and S5, constructing the crop root system microbiome. By means of the method, a large number of bacteria can be separated and cultured in a short time with less manpower, the types of the bacteria can be rapidly identified through a one-key sequence analysis process, crop root microbiome can be assembled, and technical support is provided for obtaining more crop root microbial resources.

Description

technical field [0001] The invention relates to a method for high-throughput separation and cultivation of crop root microbiome in the field of biotechnology. Background technique [0002] During the evolution of plants, the transition from aquatic to terrestrial lifestyles formed root tissue, which helps plants obtain nutrients and water from the soil. There are a large number and variety of microorganisms inhabiting the soil. These microorganisms are selected and enriched by the roots of plants to form root microbiomes. They accompany the entire growth cycle of plants and participate in plant growth and development, nutrient absorption, and disease resistance. physiological process. The interaction between plant and root microbiome is closely related to important agricultural issues such as efficient utilization of crop nutrients, continuous cropping, and crop rotation. [0003] The interaction between plant and root microbiome involves plant molecular biology (micro) an...

AI Technical Summary

Problems solved by technology

Although this method has greatly improved the throughput of microbial isolation and culture, it requires a lot of manpower and time in the later identification process. Up to now, this method is still not popular in any laboratory

[0005] The root microbiome contains tens of thousands of bacteria, and currently only about 1% of bacteria are isolated and cultured. The factors that limit the establishment of root microbiome resources include three aspects: 1. Isolation and culture methods. The commonly used monoclonal culture method is through solid Pick a large number of single clones from the culture medium, and separate and culture each single clone. On a culture plate, when the reproduction speed of a dominant bacterium is significantly better than other bacteria, it is likely that the single clones picked from the entire plate are all It is a kind of bacteria. In order to obtain more bacterial species, it is necessary to expand the isolation amount of the plate to increase the possibility of more bacterial species isolation. This method consumes a lot of manpower, material resources and time; 2. The sequencing method, the traditional The Sanger sequencing identification method is only suitable for the identification of pure bacteria because it cannot distinguish the bacteria in the mixed bacteria one by one

In addition, the Sanger identification method also has the disadvantages of heavy workload, easy contamination and loss of low-abundance bacteria, so it is not suitable for the identification of large batches of samples; 3 sequence analysis, after obtaining the bacterial sequence, professional bioinformatics personnel are required to analyze it. Researchers of systematic learning can only rely on the detection company to return the sequence comparison results, and cannot perform autonomous operations. While they have no control over the sequencing results, they still consume a lot of money

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