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Potassium hydrogen pump protein, and coding gene and applications thereof

A gene and protein technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2014-03-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the sodium-hydrogen pump, there are still relatively few discoveries and research reports on the potassium-hydrogen pump

Method used

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  • Potassium hydrogen pump protein, and coding gene and applications thereof
  • Potassium hydrogen pump protein, and coding gene and applications thereof
  • Potassium hydrogen pump protein, and coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Cloning of Nucleotide Sequence Encoding Potassium Hydrogen Pump

[0022] (1) Extraction and purification of total DNA of Bacillus alkalophilus (N16-5, CGMCC NO.0369)

[0023] Take 20 grams of fresh wet cells of Bacillus alkalophilus (N16-5, CGMCC NO.0369), suspend in 10 ml of 50 mmol / L Tris buffer (pH 8.0), add a small amount of lysozyme and 8 ml of 0.25 mg mol / L ethylenediaminetetraacetic acid (EDTA) (pH 8.0), mix well and place at 37°C for 20 minutes, then add 2 ml of 10% sodium dodecyl sulfate (SDS), place at 55°C for 5 minutes, use Equal volumes of phenol and chloroform were extracted once respectively, and the supernatant solution of the last extraction was taken, and 2 times the volume of ethanol was added to precipitate DNA. After the DNA recovered from the precipitation was washed successively with 70 volume % ethanol solution and absolute ethanol, the obtained DNA was dissolved in 0.5 ml TE buffer solution (pH 8.0, 10 mmol / L Tris, 1 mmol / L EDTA), and 10 3 mic...

Embodiment 2

[0026] Example 2 Verification of the gene function encoding the potassium-hydrogen pump

[0027] (1) Construction of recombinant cloning vector pUC18-BspN165-cha

[0028] The PCR product obtained above was purified using Cycle-pure Kit. The purified PCR product was ligated with pUC18 using Fast clone Kit, and the constructed recombinant vector pUC18-Bspn165-cha was introduced into Escherichia coli K12BW25113 by chemical transformation. The positive clone Escherichia coli K12 (pUC18-Bspn165-cha) containing the recombinant vector pUC18-Bspn165-cha was obtained by screening on the LB plate containing 100 μg / ml ampicillin and PCR verification, and the amplification of the pUC18-Bspn165-cha insertion was confirmed by sequencing. The augmented sequence is completely consistent with the nucleotide sequence of the potassium hydrogen pump.

[0029] (2) Construction of Escherichia coli K12 BW25113 potassium hydrogen pump deletion body

[0030]By designing primers containing the DNA f...

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PUM

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Abstract

The invention relates to a potassium hydrogen pump protein with an amino acid sequence represented by SEQ ID No.2. Or, the potassium hydrogen pump protein has an amino acid sequence with potassium hydrogen pump protein activity, wherein the sequence is obtained by that the amino acid sequence represented SEQ ID No.2 is subjected to substitution, deletion, or addition of one or more amino acid residues. The invention also relates to a coding gene of the potassium hydrogen pump protein. The gene has a nucleotide sequence represented by SEQ ID No.1, or the gene has a nucleotide sequence coding the amino acid sequence represented by the SEQ ID No.2. The invention also relates to a recombinant vector and cells comprising the potassium hydrogen pump gene, and an application of the potassium hydrogen pump protein, the coding gene thereof, and the recombinant vector and the cell comprising the gene in culturing alkali-resistant genetically modified organisms.

Description

technical field [0001] The present invention relates to a potassium-hydrogen pump protein, and also relates to a potassium-hydrogen pump protein coding gene, a recombinant vector and a cell containing the potassium-hydrogen pump gene, a potassium-hydrogen pump protein and a recombinant vector containing the gene and a cell in the potassium-hydrogen pump protein and its coding gene. Application in breeding transgenic organisms with alkali tolerance. Background technique [0002] Potassium hydrogen pump (K + / H + antiporter) is responsible for K + / H + A transmembrane transport protein of exchange, widely present in the plasma membrane of bacteria, human and higher plants and the membrane of many eukaryotic organelles. plasma membrane H + -ATPase uses the energy of splitting ATP to convert H + pumps the cell out of the cytoplasm, producing H across the plasma membrane + The electrochemical potential gradient provides energy to drive the potassium-hydrogen pump protein o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/32C12N15/31C12N15/63C12N1/21C12N5/10A01H5/00C12R1/07C12R1/19C12R1/125
Inventor 马延和翟磊薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI