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Genetically modified efficient phosphate solubilizing engineering bacterial strain and application thereof

A technology of engineering bacteria and genetic modification, applied in the direction of application, microbial-based methods, bacteria, etc., can solve the problems of low frequency of beneficial mutations, no genetic modification of phosphate-solubilizing bacteria, difficulty in effectively controlling the direction and nature of mutations, etc.

Active Publication Date: 2014-02-12
山东禹城瑞利源科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the obtained engineered bacteria use physical mutagenesis technology, which on the one hand produces a low frequency of beneficial mutations, and on the other hand it is difficult to effectively control the direction and nature of the mutation
In addition, the invention does not involve the genetic modification of phosphate-solubilizing bacteria using genetic engineering technology

Method used

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  • Genetically modified efficient phosphate solubilizing engineering bacterial strain and application thereof
  • Genetically modified efficient phosphate solubilizing engineering bacterial strain and application thereof
  • Genetically modified efficient phosphate solubilizing engineering bacterial strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1, construction of highly efficient phosphorus-solubilizing engineering bacteria PS57H1

[0049] The construction of highly efficient phosphorus-solubilizing engineering bacteria, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) PS57H1CGMCC No.6089, the specific method includes the following steps:

[0050] 1. Screening of phosphobacteria PS57 and identification of 16S rDNA

[0051] 1. Primary screening

[0052] The screened items were the rhizosphere soils of crops or horticultural plants from Xiaotangshan in Beijing, Hefei in Anhui, Langfang in Hebei, Yichang in Hubei, Yantai in Shandong, Jiadengling in Xinjiang, and Nanyang in Henan. The specific screening method is as follows: Weigh 5g of each soil sample, put them into Erlenmeyer flasks filled with 100mL sterile water and glass beads, vibrate vigorously on a shaker for 30min, then keep in a water bath at 80°C for 15-20min. Then serially diluted with sterile water to 10 -3 、10 -4 、10 -5 、10 -6 、...

Embodiment 2

[0102] Example 2. Detection of Phosphorus Solubilizing Ability of Bacillus amyloliquefaciens PS57H1 CGMCC No.6089 (Phosphorus Solubilizing Ability of Inorganic Phosphorus)

[0103] 1) Qualitative determination of plates

[0104] Scrape off the bacterial lawn after activating the strains to be tested, and add an appropriate amount of sterile water to make a uniform bacterial suspension (the bacterial concentration is CFU (unit / ml) = 1.84×10 9 ), use a sterilized dropper to drip the bacterial suspension into the bacterial screening medium containing inorganic phosphorus (the formula is: glucose 10g, tricalcium phosphate 5g, ammonium sulfate 0.5g, sodium chloride 0.2g, magnesium sulfate 0.1g , Potassium chloride 0.2g, yeast powder 0.5g, manganese sulfate 0.002g, 0.4% bromophenol blue (pH6.7) 6mL, agar 18g, distilled water 1000mL, pH7.0-7.5, the culture medium was prepared by conventional methods before use 115°C for 30min), evenly drop 4 spots on the solid culture dish, transfer...

Embodiment 3

[0111] Embodiment 3, preparation highly effective phosphate-dissolving microbial bacterial agent

[0112] The fermentation process of Bacillus amyloliquefaciens PS57H1 CGMCC No.6089:

[0113] 1) Prepare inorganic phosphorus bacterial liquid medium, the formula is: glucose 10g, ammonium sulfate 0.5g, yeast powder 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, sulfuric acid Manganese 0.03g, calcium phosphate 5g, distilled water 1000mL, pH 7.0-7.5, the medium was sterilized at 115°C for 30min by conventional methods before use;

[0114] 2) Inoculate Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) PS57H1 CGMCC No.6089 at a ratio of 5% in the liquid medium for inorganic phosphorus bacteria in step 1), and culture it for 20-24 hours at a shaker flask rotating speed of 180-200r / min to obtain the fermented bacterial liquid of Bacillus phospholyticus PS57H1, and obtain a solid microbial agent after drying.

[0115] The acti...

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Abstract

The invention discloses a genetically modified efficient phosphate solubilizing engineering bacterial strain and an application thereof. The bacterial strain is bacillus amyloliquefaciens PS57H1 CGMCC No.6089, phosphate solubilizing bacteria PS57 are screened and obtained from plant rhizosphere soil to construct a complete expressing element containing glucose dehydrogenase (gdh) genes, and then the gene expressing element is electro-transformed into host bacteria PS57 to obtain the bacterial strain. According to experiments, the content of dissoluble phosphorus in a culture medium can reach up to 58.8mg / L when the bacterial strain is cultured in the inorganic phosphorus culture medium (main phosphorus is from indissoluble calcium phosphate with the phosphorus content of 5g / L) for 72 hours, so that the bacterial strain has high phosphate solubilizing effect and can be used as phosphate solubilizing bacteria to be applied to microbial fertilizer production.

Description

technical field [0001] The invention relates to a high-efficiency phosphorus-dissolving bacterium modified by genetic engineering technology (the meaning of modification is the same as transformation) in the technical field of microorganisms and genetic engineering and its application in the preparation of microbial fertilizers in biological fertilizers. Background technique [0002] Phosphorus is one of the main nutrient elements that limit plant growth, and it exists mainly in the form of insoluble minerals in soil. Phosphorus-soluble microorganisms play an important role in biological systems related to the soil phosphorus cycle. They can convert insoluble inorganic phosphorus into soluble phosphorus (such as calcium monohydrogen phosphate, etc.), and improve the utilization rate of phosphorus by crops. However, the number of phosphorus-solubilizing microorganisms in the root circle of plants is usually insufficient to exert their effectiveness. Therefore, it is necessar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C05G3/00C12R1/07
Inventor 孙立文许仁杰李庆财赵学民
Owner 山东禹城瑞利源科技有限公司
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