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Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene

A Thiothiobacillus, expression level technology, applied in the field of molecular biology

Inactive Publication Date: 2016-09-07
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The research on acidophilic Thiobacillus thiooxidans has never stopped, but its biochemical process needs to be further supplemented and perfected

Method used

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  • Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene
  • Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene
  • Specific primers for detection of mRNA expression level of Acidithiobacillus thiooxidans sor gene

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Embodiment 1

[0026] Example 1. Screening and acquisition of specific primer pairs

[0027] sulfur oxidoreductase by sor Encoded by the gene, this enzyme has the ability to catalyze the disproportionation reaction of elemental sulfur, which is crucial in the oxidation process of elemental sulfur and reduced inorganic sulfur compounds. Sulfur oxidoreductase exists in some thermophilic archaea and acidophilus, and it has been reported in acidophilic thermophilic archaea such as Tengchong acidophilus and Brucella acidophilus and acidophilic bacteria such as thermophilic Thiobacillus .

[0028] Through whole genome sequencing, we obtain Acidithiobacillus thiooxidans The whole genome sequence information of A01, and the existence of the gene encoding sulfur oxidoreductase was reported for the first time in this species. according to A. thiooxidans A01 sor Gene (NCBI accession number is KJ483962), multiple sets of primers were designed through Primer 5, trying to meet the rules of primer ...

Embodiment 2

[0040] Example 2, the expression level of sulfur oxidoreductase gene mRNA of acidophilus thiooxidans under different energy sources

[0041] 1. Culture medium and culture conditions

[0042] strain Acidithiobacillus thiooxidansA01 was cultured in 9K medium and medium 71 of German Biomaterials Resource Center (DSMZ medium 71) respectively. Among them, 9K medium containing 10.0 g / L elemental sulfur was used to adjust the initial pH of the culture medium to 2.0 with 5 mol / L H2SO4, while the pH of DSMZ medium 71 medium containing 5.0 g / L sodium thiosulfate was adjusted to 4.4. Bacteria were cultured in conical flasks (250 mL) filled with 100 mL of culture medium, and the initial concentration of bacteria was 2.5×10 6 cells / ml, the culture temperature was 30°C, and the shaking frequency was 170 rpm.

[0043] 2. Extraction of RNA

[0044] In the mid-logarithmic phase (54 h) (average of three repeated experiments) the bacterial cells were recovered by centrifugation and used to...

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Abstract

The invention provides specific primers for detection of mRNA expression level of an Acidithiobacillus thiooxidans sor gene. The nucleotide sequences are as follows: an upstream primer sequence of 5'-AAGCCCGTGCCTAAAGTG-3', and a downstream primer sequence of 5'-CTGCCATAGTTGGTGTTGT-3'. The specific primers can detect the transcription level of Acidithiobacillus thiooxidans sor gene, and hace the advantages of high sensitivity and good specificity in detecting mRNA gene expression level. Through the real-time monitoring of mRNA expression level of sor gene in sulfur oxidation process, the primers are used to explain the sulfur oxidation mechanism of Acidithiobacillus thiooxidans.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a method for detecting acidophilic Thiobacillus thiooxidans sor Specific primers for gene mRNA expression levels. Background technique [0002] As an inorganic chemoautotrophic, Gram-negative, rod-shaped extremophile, the bioleaching of Thiobacillus acidophilus and sulfide minerals (coal mine, chalcopyrite, pyrite, chalcocite, etc.) Closely related, it has broad prospects in the field of industrial applications. Thiobacillus acidophilus thiooxidans can use elemental sulfur and reduced inorganic sulfur compounds (Reduced Inorganic Sulfur Compounds, RISCs for short) as energy substances. The research on acidophilic Thiobacillus thiooxidans has never stopped, but its biochemical process needs to be further supplemented and perfected. Under the action of the sulfur oxidase system in Thiobacillus acidophilus thiooxidans, elemental sulfur or reduced inorganic sulfur compounds unde...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2600/178C12Q2531/113C12Q2563/107
Inventor 尹华群
Owner CENT SOUTH UNIV
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