Mytilus coruscus multi-drug resistance protein Abcc -- novel marine biological pollution detection marker

A multi-drug resistant, thick-shelled mussel technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA preparation, etc., can solve problems such as pollution, achieve specificity, improve fidelity and activity, specific effect

Inactive Publication Date: 2019-02-15
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

There is no report on the use of the multidrug resistance protein Abcc of thick shelled mussels as a new marine biofouling detection marker

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The multidrug resistance protein Abcc of the thick shell mussel, the multidrug resistance protein Abcc is a cloned gene, obtained by cloning the gene core fragment of the thick shell mussel Abcc by gene cloning technology.

[0028] The cloning steps of the Abcc gene are:

[0029] 1) Extraction of total RNA: extract total RNA from each tissue using thick-shelled mussels as material;

[0030] 2) Synthesis of the first strand of cDNA: using the total RNA sample obtained in step 1 as a template, use the M-MLV reverse transcription kit to perform reverse transcription synthesis of cDNA to obtain the first strand of cDNA;

[0031] 3) PCR amplification of the core fragment of the multidrug resistance protein Abcc: According to the sequence of the multidrug resistance protein Abcc of thick shell mussels in the NCBI database, the conserved region of the amino acid sequence was analyzed, and degenerate primers were designed using primer5.0 software to amplify the The core sequen...

Embodiment 2

[0043] The multidrug resistance protein Abcc of thick shell mussel, the cloning steps are as follows:

[0044] 1) Extraction of total RNA: extract total RNA from each tissue using thick-shelled mussels as material;

[0045] Take a RNase-free 2ml EP tube, add 500ul Trizol to the tip of the pipette used, pick up the brain tissue sample with tweezers soaked in alcohol and dissolve it in the Trizol liquid, grind it with an electric homogenizer until the tissue is completely dissolved in the liquid, and fill up the Trizol Centrifuge at 1mL, 4°C, 12000rpm, 10min, take the supernatant and put it into a 1.5mL EP tube. Impurities cannot be absorbed, then add 200ul of chloroform, shake vigorously, let stand at room temperature for 5 minutes, centrifuge at 4°C, 12000rpm, 15min , take the uppermost liquid of the three layers into a new 1.5mL EP tube, add 500ul of isopropanol, room temperature for 10min or -20°C overnight, then centrifuge at 4°C, 12000rpm for 10min, remove the supernatant,...

Embodiment 3

[0061] The 15-day-old thick-shelled mussel was used as the biological sample for monitoring water pollution, and the thick-shelled mussel was divided into two groups, which were divided into the non-pollution control group and the water area group to be tested. Breeding in the water body of the test water, 3 parallels in each group, after 7 days of feeding, 3 samples were taken in each parallel for measurement, 3x3=9 data were measured in each group, and the 9 data in each group were mathematically counted to obtain each group. Bei's results.

[0062] The relative expression levels of the mussel multidrug resistance protein Abcc in digestive glands and gills were measured using the steps in Example 2. Test results: the relative expression values ​​of the control group in the digestive gland and gills were 2.34 and 1.97, and the relative expression values ​​of the test group in the digestive gland were 1.99 and 1.52. The results showed that the relative expression value of the ...

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Abstract

The invention discloses a mytilus coruscus multi-drug resistance protein Abcc. The multi-drug resistance protein Abcc is a cloned gene, which is obtained by cloning a gene core fragment of mytilus coruscus Abcc by a gene cloning technology. The invention also discloses a novel marine biological pollution detection marker, which uses mytilus coruscus as a biological sample for detecting marine pollution, and uses the mytilus coruscus multi-drug resistance protein Abcc as a detection marker for marine pollution. The relative expression amount of a multi-drug resistance protein Abcc gene of the mytilus coruscus multi-drug resistance protein Abcc is the highest in digestive glands and gills, and a PCR reaction system for cloning can ensure the specificity of an amplification reaction and improve the PCR amplification efficiency. The detection marker provided by the invention has strong specificity, can directly take target cells or target molecules in organisms as reaction endpoints to carry out sensitive biological reactions at the molecular level, and has an early warning effect on marine pollutant exposure and toxicity effects.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the multidrug resistance protein Abcc of thick-shelled mussels, a novel detection marker for marine biological pollution. technical background [0002] In recent years, with the rapid development of industry and agriculture, the coastal waters have been polluted to varying degrees, especially the development of the petroleum industry and marine transportation, making oil the most important pollutant in the coastal waters. Oil pollution can not only make fish, shrimp, The taste of shellfish and seafood changes, which can produce toxic effects in severe cases, causing serious harm to the marine ecological environment and fishery resources; at the same time, because heavy metals are easy to accumulate in the bottom and are not easy to degrade, they are often bio-accumulated and have potential harm to human health. The threat of heavy metal pollution is also one of the envi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/10C12Q1/6876
CPCC07K14/43504C12Q1/6876C12Q2600/158
Inventor 祁鹏志董文强任世太郭宝英
Owner ZHEJIANG OCEAN UNIV
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