K<+>/H<+>antiporter protein, its coding gene, and applications of protein and coding gene

A protein and gene technology, applied in the field of coding genes of potassium hydrogen pump protein

Inactive Publication Date: 2013-05-08
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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  • K&lt;+&gt;/H&lt;+&gt;antiporter protein, its coding gene, and applications of protein and coding gene
  • K&lt;+&gt;/H&lt;+&gt;antiporter protein, its coding gene, and applications of protein and coding gene
  • K&lt;+&gt;/H&lt;+&gt;antiporter protein, its coding gene, and applications of protein and coding gene

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 pseudofirmus OF4

[0023] Take 20 grams of fresh wet bacteria of Bacillus pseudofirmus OF4, suspend in 10 ml of 50 mmol / L Tris buffer solution (pH 8.0), add a small amount of lysozyme and 8 ml of 0.25 mmol / L ethylene glycol Eminetetraacetic 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, add equal volumes of phenol and chloroform respectively Each extraction was performed once, 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 microliter...

Embodiment 2

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

[0027] (1) Construction of recombinant cloning vector pUC18-BpOF4-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-BpOF4-cha was introduced into Escherichia coli K12BW25113 by chemical transformation. The positive clone Escherichia coli K12 (pUC18-BpOF4-cha) containing the recombinant vector pUC18-BpOF4-cha was obtained by screening on the LB plate containing 100 μg / ml ampicillin and PCR verification, and the amplification of the pUC18-BpOF4-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 K12BW25113 potassium hydrogen pump deletion body

[0030]By designing primers containing the DNA fragments of...

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Abstract

The invention relates to a K<+> / H<+>antiporter protein. The K<+> / H<+>antiporter protein has an amino acid sequence represented by SEQ ID No:2, or has an amino acid sequence obtained through substituting, deleting or adding one or more amino acid residues to the amino acid sequence represented by the SEQ ID No:2 and still having K<+> / H<+>antiporter protein activities. The invention also relates to a coding gene of the K<+> / H<+>antiporter protein. The gene has a nucleotide sequence represented by SEQ ID No:1, or has a nucleotide sequence coding the amino acid sequence represented by SEQ ID No:2. The invention further relates to a recombinant vector containing the K<+> / H<+>antiporter gene, a cell containing the K<+> / H<+>antiporter gene, and applications of the K<+> / H<+>antiporter protein, the coding gene thereof, the recombinant vector containing the gene and the cell containing the gene in the cultivation of alkali-resistant transgenic 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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IPC IPC(8): C07K14/32C12N15/31C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 马延和翟磊薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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