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Phosphate solubilizing gene for promoting organic acid secretion

A phosphorus-dissolving gene and gene technology, applied in the field of phosphorus-dissolving genes that promote the secretion of organic acids

Inactive Publication Date: 2013-11-27
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

There is no report that genes or proteins with higher homology with the present invention have the function of promoting the host bacteria to produce organic acids in large quantities

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The cDNA full-length library construction of embodiment 1 Penicillium oxalicum

[0033] The strain Penicillium oxalicum strain I1 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 cultivated on an insoluble phosphorus inorganic salt medium for 3 days, and about 0.2 g of fresh mycelium was taken ( Such as figure 1 ), ground with liquid nitrogen, extracted total RNA with RNAiso plus kit, took 1 μg RNA as a template for the synthesis of the first strand of cDNA, and reverse transcribed by SMARTscriptTM under the guidance of 3'SMART CDS Primer IIA and SMARTer IIA oligonucleotide primers 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 p...

Embodiment 2

[0034] Example 2 Screening and Bioinformatics Analysis of Phosphorus-Solubilizing Genes

[0035] Dilute the bacterial culture solution of the library onto the insoluble phosphorus medium containing ampicillin (100 μg / mL), incubate at 37°C for 3-4 days, and observe whether there is a phosphorus-dissolving transparent circle. The results are as follows: image 3 As shown, some strains produced transparent circles on the insoluble phosphorus inorganic salt medium, and the transformants that produced transparent circles were transferred to culture to determine their stability, and the functionally stable colonies were extracted from the plasmids and sequenced to obtain DNA sequences. Blast was used for comparison analysis, and DNAMAN6.0 was used for open reading frame analysis, and the translated proteins were compared and analyzed on the PDB database.

Embodiment 3

[0036] Construction of embodiment 3 expression vector, transformation host, positive clone identification

[0037] Primer design (Forward: 5'-TATTCGGAATTCATGACAGACCTCAAGTCCAT-3'; Reverse: 5'-CAAGTACTCGAGTTAAACGATAGTCTCTAGCTC-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 pET28a(+), and the expression vector pET28a(+) and restriction site sequence were 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 follows Figur...

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Abstract

The invention provides a phosphate solubilizing gene psg I-13. The nucleotide sequence of the phosphate solubilizing gene psg I-13 is as shown in SEQ ID No.1. The nucleotide sequence of a protein coded by the phosphate solubilizing gene psg I-13 is as shown in SEQ ID No.2. The phosphate solubilizing gene psg I-13 cloned through heterologous expression can be used for efficiently generating organic 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 organic acids. 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, organic acids, 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 ba...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C12N1/21C12N15/11C12P7/54C12P7/40C12P7/46C12P7/50C07K14/385C09K17/14C09K101/00
Inventor 龚明波
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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