Construction method of compound microbial agent

A technology of compound microbial agent and construction method, which is applied in the field of synthetic biology and biological fertilizer science, can solve the problems of single function of microbial fertilizer, inability to reflect rational design principles, unstable field effect, etc.

Pending Publication Date: 2022-01-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve problems such as the single function of a single strain of microbial fertilizer, unstable field effect and low efficiency of the bottom-up (Bottom-up) method, which cannot reflect rational design principles, the first object of the present invention is to provide a composite microorganism The construction method of inoculum; the second purpose is to provide the composite microbial inoculum constructed by the above construction method; the third purpose is to provide the application of the above-mentioned composite microbial inoculum as microbial fertilizer in actual production

Method used

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  • Construction method of compound microbial agent
  • Construction method of compound microbial agent
  • Construction method of compound microbial agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Reconstructing the genome-scale metabolic model of each strain

[0053] The genome of each strain in the experiment was sequenced to obtain the complete map of each strain. Reconstruct the draft genome by automatic (ModelSEED) or semi-automatic means; manually optimize the reconstructed draft genome, determine and verify the substrate and coenzyme, the molecular formula and charge of the metabolite, the stoichiometric coefficient and directionality of the reaction, and determine the gene-protein reaction relationship , add metabolite identifiers, add spontaneous reactions, transport reactions and exchange reactions, determine biomass composition; transform the reconstructed model into a mathematical model, initialize COBRA Toolbox, load the reconstructed model into MATLAB, verify the stoichiometric matrix S and set Objective functions and constraints; debug models, check model mass balance charge balance, make corrections for unbalanced reactions, test chemic...

Embodiment 2

[0056] Example 2: Siderophilic production ability of each bacterial strain

[0057] Prepare CAS blue detection solution solution, A: Dissolve 60.5mg CAS in 50mL deionized water, add 10mL FeCl 3 solution (containing 1mM FeCl 3 ·6H 2 O, 10mM HCl); solution B: dissolve 72.9mg HDTMA (hexadecyltrimethylammonium bromide) in 40mL deionized water; slowly inject solution A into solution B along the beaker wall to obtain CAS blue color test solution.

[0058] After the supernatant of the culture medium of the strain was sterilized, it was mixed with an equal volume of the CAS detection solution, fully reacted, and the OD was measured 630 , get the absorbance value As, adjust to zero with double distilled water as the control; mix the uninoculated culture medium with the CAS detection solution in equal volume, and measure the OD 630 , to get the absorbance value Ar.

[0059] results and analysis:

[0060] The As / Ar value represents the relative content of siderophiles in the sample...

Embodiment 3

[0061] Embodiment 3: Determination of phosphorus solubilizing ability of each bacterial strain

[0062] Prepare NBRIP medium according to the following formula: glucose 10g, ammonium sulfate 0.1g, potassium chloride 0.2g, calcium phosphate 5g, magnesium chloride hexahydrate 5g, magnesium sulfate heptahydrate 0.25g, distilled water to 1000mL, pH 7.0.

[0063] Activate each strain in LB medium, insert the strain to be tested into phosphate liquid medium (NBRIP), and use no bacteria as blank control, 28°C, 170r min -1 Under the condition of shaking culture for 7 days, the molybdenum antimony anti-colorimetric method was used to quantitatively measure the dissolved phosphorus content and the corresponding pH value.

[0064] results and analysis:

[0065] Such as image 3 It was shown that Pseudomonas fluorescens Pse_flu and Bacillus megaterium Bac_meg had higher ability to solubilize phosphorus.

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Abstract

The invention discloses a construction method of a compound microbial agent. According to the method, a constraint-based metabolic model reconstruction method in synthetic biology is utilized to predict a potential interaction relationship, the growth promoting relationship of all strains is evaluated, the antagonism relationship between the strains is evaluated through a streak confrontation experiment, the carbon source utilization spectrum of all the strains is measured, and the ecological measure is calculated. And finally, quantitative PCR detection is performed on the dynamic change of each member of the flora under the plant rhizosphere condition, and the growth promoting effect at the same time is verified to obtain the compound microbial agent. The obtained composite flora is stable in structure in a rhizosphere in-situ environment and has an obvious growth promoting effect.

Description

technical field [0001] The invention relates to a construction method of a composite microbial bacterial agent, the composite microbial bacterial agent obtained by the construction method and an application thereof, belonging to the fields of synthetic biology and biological fertilizer science. Background technique [0002] Bio-fertilizers have played a vital role in my country's soil fertilization and improvement, crop quality and yield increase, chemical fertilizer reduction and efficiency increase, and resource recycling; in the national green agricultural development and rural revitalization plans and other strategies, the basic research of bio-fertilizers , application innovation and industrial development have become key development goals in the new era. After nearly 20 years of steady and rapid development of my country's bio-fertilizer industry, there are currently more than 9,200 microbial fertilizer products registered by the Ministry of Agriculture and Rural Affair...

Claims

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Application Information

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IPC IPC(8): C12N1/20G16B5/00A01N63/27A01N63/20A01N63/22A01P21/00C12R1/38C12R1/07C12R1/39C12R1/11C12R1/01
CPCC12N1/20G16B5/00A01N63/27A01N63/20A01N63/22Y02W30/40
Inventor 张瑞福王伟许欢欢李森徐志辉张楠荀卫兵缪有志沈其荣
Owner NANJING AGRICULTURAL UNIVERSITY
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