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Application of acid phosphatase and related biological materials thereof in constructing phosphate-solubilizing engineering bacteria

A phosphohydrolase and phosphorus-dissolving technology, applied in microorganism-based methods, applications, microorganisms, etc., can solve the problems of many by-products and difficult purification.

Active Publication Date: 2016-07-20
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are mainly two methods to produce nucleotides. One is chemical synthesis, which uses bacterial fermentation to produce inosine, and then uses POCl 3 Phosphorylation, this method has many by-products and is difficult to purify; another method is to use Escherichia coli inosine kinase to phosphorylate inosine, this process requires the participation of ATP, and ATP needs to be regenerated by fermentation of Ammonogenes bacteria, which limits the enzymatic method synthetic application

Method used

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  • Application of acid phosphatase and related biological materials thereof in constructing phosphate-solubilizing engineering bacteria
  • Application of acid phosphatase and related biological materials thereof in constructing phosphate-solubilizing engineering bacteria
  • Application of acid phosphatase and related biological materials thereof in constructing phosphate-solubilizing engineering bacteria

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Effect test

Embodiment 1

[0058] Embodiment 1, preparation and functional verification of acid phosphatase BmacpA

[0059] 1. Cloning of BmacpA gene and BmacpB gene

[0060] Genomic DNA of Bacillus megaterium ACCC10010 and Bacillus megaterium ACCC02970 were extracted respectively, using the genomic DNA of Bacillus megaterium ACCC10010 as a template, using P1 and P2 as primers, PCR amplified the BmacpA gene; using the genomic DNA of Bacillus megaterium ACCC02970 as a template, Using P3 and P4 as primers, PCR amplified BmacpB gene. Among them, the sequences of P1, P2, P3 and P4 are as follows: P1: 5′-ATGTATGTGAAACGATATCG-3′, P2: 5′-CTACTTTTTGTCGAACACATA-3′, P3: 5′-ATGGTAAATCGCACTACAAA-3′, P4: 5′-CTATTTTTGGTTATATAAGC-3 '.

[0061] The obtained BmacpA gene PCR product and the BmacpB gene PCR product are subjected to electrophoresis respectively, and the results show that the BmacpA gene PCR product and the BmacpB gene PCR product are about 600bp bands, and the BmacpA gene PCR product and the BmacpB gene ...

Embodiment 2

[0106] Embodiment 2, the cultivation and functional identification of acid phosphatase phosphate-dissolving engineering bacteria

[0107] 1. Construction of Bacillus megaterium acid phosphatase BmacpA gene shuttle expression vector

[0108] In order to obtain phosphate-soluble engineering bacteria that highly express Bacillus megaterium acid phosphatase BmacpA gene, it is necessary to first construct a shuttle expression vector that can express across hosts. pHT43 (product of German MoBiTec company, purchased from Wuhan Miaoling Biotechnology Co., Ltd.) is a commonly used shuttle expression vector, carrying BamHI and XbaI enzyme recognition sites, with ampicillin and chloramphenicol resistance genes, induced by IPTG, can highly Level secreted expression of target protein. DNAMAN software was used to analyze the gene sequence of Bacillus megaterium acid phosphatase BmacpA, and it was found that there were no restriction sites of BamHI and XbaI, and the recombinant plasmid expr...

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Abstract

The invention discloses application of acid phosphatase and related biological materials thereof in constructing phosphate-solubilizing engineering bacteria. The application refers to application of protein as shown in a) or b) or c) or d) in phosphohydrolase: a) protein constituted by amino acid sequences on 1st-203rd sites of SEQ ID N0.2; b) protein constituted by amino acid sequences on 26th-203rd sites of SEQ ID N0.2; c) fusion protein obtained by fusing a protein tag / protein tags on carboxyl terminal (C terminal) or / and amino terminal (N terminal) of the protein of a) or b); and d) protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of an amino acid sequence as shown in SEQ ID No.2 or SEQ ID No.6 and has the activity of the acid phosphatase. According to the application disclosed by the invention, biological engineering bacteria, which can effectively activate soil phosphorus nutrients, can be cultivated.

Description

technical field [0001] The invention relates to the use of acid phosphatase and related biological materials in the biological field, in particular to the application of acid phosphatase and related biological materials in building phosphorus-dissolving engineering bacteria. Background technique [0002] Phosphorus is one of the three essential elements for plant growth and development, and plays an important role in the life process. The phosphorus used by plants mainly comes from the soil. At present, 2 / 3 of the cultivated land in my country is deficient in phosphorus. The main reason for phosphorus deficiency is the insufficient content of available phosphorus in the soil. About 95% of phosphorus is insoluble and ineffective phosphorus, which is difficult for plants to absorb and utilize. my country consumes about 10.5-12 million tons of phosphate fertilizers every year (2010 China Chemical Industry Information Network), but the plant utilization rate of phosphate fertil...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N1/21C12R1/19C12R1/01
CPCC12N9/16C12N15/70C12N15/74C12Y301/03002
Inventor 孙静文周卫程明芳李书田王玉军
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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