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Phosphate solubilizing gene for promoting acetic acid secretion

A phosphorus-dissolving gene and gene technology, applied in the field of phosphorus-dissolving genes that promote the secretion of acetic acid, can solve the problem of no functional analysis and the like

Inactive Publication Date: 2013-11-27
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Other reports are hypothetical proteins without functional analysis

Method used

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  • Phosphate solubilizing gene for promoting acetic acid secretion
  • Phosphate solubilizing gene for promoting acetic acid secretion
  • Phosphate solubilizing gene for promoting acetic acid secretion

Examples

Experimental program
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Effect test

Embodiment 1

[0033] The cDNA full-length library construction of embodiment 1 Aspergillus niger

[0034] The strain Aspergillus niger strain H1 used in the experiment was screened from soil samples by the Agricultural Strain Collection Center of the Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences. It was cultured on the insoluble phosphorus inorganic salt medium for 3 days, and about 0.2 g of fresh mycelia (such as figure 1 ), ground with liquid nitrogen, extracted total RNA with RNAisoplus kit, took 1 μg of RNA as a template for synthesizing the first strand of cDNA, guided by 3'SMART CDS Primer IIA and SMARTer IIA oligonucleotide primers, passed SMARTscriptTM reverse transcriptase The first-strand cDNA was synthesized by reverse transcription at 70°C for 2 min; double-stranded cDNA was synthesized by LD-PCR using 2 μL of the first-strand cDNA product as a template. Use CHROMA SPIN+TE-1000 to purify cDNA, remove fragments sm...

Embodiment 2

[0035] Example 2 Screening and Bioinformatics Analysis of Phosphorus-Solubilizing Genes

[0036] Dilute the culture solution containing the bacteria in the library onto the insoluble phosphate medium containing ampicillin (100 μg / mL), incubate at 37°C for 3-4 days, observe whether there is a phosphate-dissolving transparent circle, and transfer the transformant that produces the transparent circle to cultivated, such as image 3 As shown, the screened clones can still produce transparent circles on the insoluble phosphorus inorganic medium after transfer, indicating that their functions are stable. The stable colonies were extracted from the plasmids and sequenced to obtain DNA sequences, which were compared by Blast on NCBI Analysis, and use DNAMAN6.0 to analyze the open reading frame, and compare and analyze the translated protein on the PDB database.

Embodiment 3

[0037] Construction of embodiment 3 expression vector, transformation host, positive clone identification

[0038] Primer design (Forward: 5'-TATTCGGAATTCATGTCTGCCAATCGAGCAAG-3'; Reverse: 5'-CAAGTACTCGAGCTACGGCTCGCCGAGGAGCCG-3'), using high-fidelity Taq enzyme to amplify the ORF of the above gene, reaction conditions: 94°C for 5min; 94°C for 1min, 60°C for 1min , 72°C 1min cycle 35 times 72°C 10min, 4°C forever. After double digestion with restriction endonucleases EcoRI and XhoI, it was connected to the expression vector pGEX-6p. The sequence of the restriction site and the map of the expression vector pGEX-6p are as follows: Figure 4 , Figure 5As shown, E.coli HST08 was transformed by electric shock method, and the clones that could produce transparent circles were screened on the insoluble phosphorus inorganic salt medium ( Figure 6 ), and the fragment size of the positively transformed clones was selected by PCR identification, and the amplification results were as fo...

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Abstract

The invention provides a phosphate solubilizing gene psa A. The nucleotide sequence of the phosphate solubilizing gene psa A is as shown in SEQ ID No.1. The nucleotide sequence of a protein coded by the phosphate solubilizing gene psa A is as shown in SEQ ID No.2. The phosphate solubilizing gene psa A cloned through heterologous expression can be used for efficiently generating acetic acids, has a function of converting indissoluble phosphate into effective phosphate and provides a base material for increasing the utilization rate of a phosphate fertilizer by using a molecular biology means.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a phosphorus-dissolving gene that promotes the secretion of acetic acid. Background technique [0002] The production of organic acid is a major way for phosphorus-dissolving microorganisms to release soil insoluble phosphorus. Organic acid chelates with iron, aluminum, calcium and other ions in the soil, thereby converting insoluble phosphorus into available phosphorus and improving the utilization rate of phosphate fertilizer. The common organic acids produced by phosphorus-dissolving microorganisms are succinic acid, citric acid, α-ketoglutaric acid, malic acid, pyruvic acid, lactic acid, acetic acid, formic acid, propionic acid, fumaric acid and oxalic acid. However, the application of phosphorus-dissolving microorganisms is affected by the degradation of strains, crop types, soil characteristics, climatic conditions, and the interaction between phosphorus-dissolving bacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/38C12N15/70C12N1/21C12N15/11C12P7/54C09K17/00C12R1/685C09K101/00
Inventor 龚明波
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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