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Gobiocypris rarus alpha type Na+/K+-ATPase, and encoding gene and application thereof
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A technology of rare gobi, DNA molecules, applied in the field of molecular biology and environmental biology
Inactive Publication Date: 2014-07-09
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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However, much work is needed in terms of molecular biology and toxicology, especially with regard to mechanisms of stress in the adrenal system
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Embodiment 1
[0042] Embodiment 1, the cloning of rare gobi carp α-type Na+ / K+-ATPase gene
[0043] 1. Extraction of total RNA and synthesis of first-strand cDNA
[0044] Brain tissue was isolated from mature individuals of rare gobi carp 10 months after hatching, quickly placed in liquid nitrogen, and ground into powder; transferred to a clean Ep tube, and the total brain tissue was extracted using Trizol reagent (Gibco BRL, Life Technologies, Gaithersburg, MA, USA). RNA; the obtained total RNA was treated with DNase I (promega, USA) to remove DNA contamination. The ultraviolet light absorption measurement value A260 / A280 of the obtained total RNA was greater than 1.8; 0.8% formaldehyde denaturing gel electrophoresis confirmed no degradation. The first strand of cDNA was synthesized using M-MLV reverse transcriptase (Promega, USA).
[0045] 2. PCR amplification and screening of recombinant bacteria
[0046] Merger primers were designed using Primer Premier 5 and synthesized by Beijing A...
Embodiment 2
[0054] Example 2, rare gobi carp α-type Na + / K + -Expression of ATPase gene in different tissues and different life stages
[0055] 1. Design specific primers according to the sequence obtained in Example 1
[0058] 2. Rare goby carp α-type Na + / K + -Expression of ATPase gene in different life stages
[0059] 1. Collection of samples
[0060] Take the rare goby embryos 2 hours, 6 hours, 12 hours, 24 hours, 72 hours after fertilization and the rare goby juveniles 1 day, 3 days, 7 days, 14 days, 21 days after hatching, and immediately freeze them in liquid nitrogen stored as samples to be tested.
[0061] 2. Rare gobi α-type Na + / K + -Expression of ATPase gene in different life stages
[0062] (1) Extract the total RNA of each sample to be tested, and reverse transcribe it into cDNA.
[0063] (2) Using the cDNA in step (1) as a template, perform PCR amplific...
[0074] Cortisol (Hydrocortisone) was purchased from Shanghai Biochemical Reagent Company. Pentachlorophenol (PCP) was purchased from Chem Service (West chester, PA, USA). Dimethyl Sulfoxide (DMSO) was purchased from Sigma (St. Louis, MO, USA).
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Abstract
The invention discloses gobiocypris rarus alpha type Na+ / K+-ATPase, and an encoding gene and application thereof. The gobiocypris rarus alpha type Na+ / K+-ATPase is shown as the following (a) or (b): (a) protein consisting of the amino acid sequence shown as the sequence 1 in the sequence table; and (b) protein formed by substituting and / or deleting and / or adding one or more amino acid residue, having Na+ / K+-ATPase activity and derived from the sequence 1. The invention also provides the encoding gene of the protein and designs a specific primer pair based on the gene. The gobiocypris rarus is cultivated in the water environment containing the adrenal hormone interferent, the expression quantity of the gobiocypris rarus alpha type Na+ / K+-ATPase gene of the gobiocypris rarus is changed, and the gobiocypris rarus alpha type Na+ / K+-ATPase gene has a dose-effect relationship, so that the specific primer can be used for auxiliarily detecting whether the water environment contains the adrenal hormone interferent or not. The method has the advantages of simplicity, convenience, quickness and accuracy.
Description
technical field [0001] The invention belongs to the fields of molecular biology and environmental biology, and relates to a rare gobi α-type Na + / K + -ATPase and its encoding gene and application, as well as the primer pair based on the encoding gene and its application in auxiliary detection of whether adrenal hormone or adrenal hormone disruptor is contained in the water environment. Background technique [0002] Na + / K + -ATPase, also known as the sodium pump, can catalyze the hydrolysis of ATP to supply energy and drive Na + and K + The opposite transport on both sides of the cell membrane maintains the membrane potential on both sides of the cell membrane, regulates the cell osmotic pressure, provides power for the absorption of nutrients, and plays an important role in the impulse conduction of nerve and muscle cells. Fish live in the water environment all their lives, and their body fluids are often not isotonic with the outside world. Therefore, osmotic pressu...
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