Aryl hydrocarbon receptor nuclear translocator (ARNT)-novel marine organism pollution detection marker

Abstract

The invention discloses an aryl hydrocarbon receptor nuclear translocator (ARNT). The nucleotide sequence of the gene of the aryl hydrocarbon receptor nuclear translocator is SEQ ID NO.1, and the amino acid sequence of the aryl hydrocarbon receptor nuclear translocator is SEQ ID NO.2. The invention also discloses a mytilus coruscus ARNT as a novel marine organism pollution detection marker, and amethod for detecting marine organism pollution. The ARNT disclosed by the invention has the beneficial effects that the mytilus coruscus ARNT is obtained through a gene clone technology for the firsttime; the relative expression amount of the mytilus coruscus ARNT is highest in the digestive glands and the gill of the mytilus coruscus; the mytilus coruscus ARNT can be used for detecting marine pollution; marker specificity is high, a target cell or a target molecule in a living body can be directly used as a reaction endpoint on an aspect of molecular level to carry out sensitive biological reaction so as to perform an early warning function on marine pollutant exposure and toxic effects.

Description

technical field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to an aromatic hydrocarbon receptor nuclear translocation protein ARNT, a novel detection marker for marine biological pollution. Background technique

[0002] Thick-shelled mussel (Mytilus coruscus) (Mytilidae) is one of the fastest growing marine bivalve molluscs, widely distributed in the Yellow Sea, the Korean Peninsula and Hokkaido, Japan. Because of its rich nutrition and delicious taste, it is widely welcomed by the large-scale farming market and popularity in China, which has the largest production of marine shellfish. In recent years, marine environmental pollution in China's waters has shown an intensifying trend. Furthermore, as a lower species, the thick-shelled mussel lacks an adaptive immune system, which induces vulnerability to external stimuli. This mussel species is hit by diseases produced by viruses, bacteria and even severe psychoactive a...

Examples

Embodiment 1

[0044] The fragment size of aryl hydrocarbon receptor nuclear translocator ARNT of mussel mussels is 2043bp, encoding a total of 681 amino acids.

[0045] The cloning method of the aromatic hydrocarbon receptor nuclear translocation protein ARNT of the thick shell mussel, including: preparing samples, extracting total RNA, synthesizing the first strand of cDNA, cloning the core sequence, cloning the full-length sequence, cloning and sequence analysis of PCR products and biological For informatics analysis, the degenerate primers for PCR amplification of the cloned core sequence are:

[0046] ARNT 01F: 5′-TGTACCGGGCCCATCAAAGCTTGGCC-3′ and

[0047] ARNT 02R: 5′-CACGTCTGACTAAGGAGGCTGAAA-3′, the length of the primers for PCR amplification of the core fragment, the difference between the length of the upstream and downstream primers is more suitable for the amplification of the PCR reaction, and the T of the upstream and downstream primers m The difference between the values ​​is ...

Embodiment 2

[0091] As a further optimization of Example 1, the cloning method of the mussel aromatic hydrocarbon receptor nuclear translocation protein ARNT specifically includes the following steps:

[0092] (1) Prepare samples:

[0093] The average individual weight of thick-shelled mussels is 60.15g, the average width of the shell under healthy conditions is 4.5cm, and the average shell height is 9.1cm. It comes from the Dongji Island Aquaculture Base (Zhoushan City, Zhejiang Province, China), and the filtered seawater is kept during the breeding period. Continue to oxygenate and cultivate at a temperature of 24-25°C and a pH value of 8.0.

[0094] (2) LPS and Cu 2+ challenge:

[0095] 1) LPS challenge: For LPS challenge, 80 adult thick-shelled mussels were randomly divided into two groups, one group of 40 individuals (control group) was injected with PBS (pH7.4) for adductor muscles and the other group of 40 individuals (Challenge group) to conduct LPS challenge. Challenged mussel...

technical field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to an aromatic hydrocarbon receptor nuclear translocation protein ARNT, a novel detection marker for marine biological pollution. Background technique

[0002] Thick-shelled mussel (Mytilus coruscus) (Mytilidae) is one of the fastest growing marine bivalve molluscs, widely distributed in the Yellow Sea, the Korean Peninsula and Hokkaido, Japan. Because of its rich nutrition and delicious taste, it is widely welcomed by the large-scale farming market and popularity in China, which has the largest production of marine shellfish. In recent years, marine environmental pollution in China's waters has shown an intensifying trend. Furthermore, as a lower species, the thick-shelled mussel lacks an adaptive immune system, which induces vulnerability to external stimuli. This mussel species is hit by diseases produced by viruses, bacteria and even severe psychoactive a...

Examples

Embodiment 1

[0044] The fragment size of aryl hydrocarbon receptor nuclear translocator ARNT of mussel mussels is 2043bp, encoding a total of 681 amino acids.

[0045] The cloning method of the aromatic hydrocarbon receptor nuclear translocation protein ARNT of the thick shell mussel, including: preparing samples, extracting total RNA, synthesizing the first strand of cDNA, cloning the core sequence, cloning the full-length sequence, cloning and sequence analysis of PCR products and biological For informatics analysis, the degenerate primers for PCR amplification of the cloned core sequence are:

[0046] ARNT 01F: 5′-TGTACCGGGCCCATCAAAGCTTGGCC-3′ and

[0047] ARNT 02R: 5′-CACGTCTGACTAAGGAGGCTGAAA-3′, the length of the primers for PCR amplification of the core fragment, the difference between the length of the upstream and downstream primers is more suitable for the amplification of the PCR reaction, and the T of the upstream and downstream primers m The difference between the values ​​is ...

Embodiment 2

[0091] As a further optimization of Example 1, the cloning method of the mussel aromatic hydrocarbon receptor nuclear translocation protein ARNT specifically includes the following steps:

[0092] (1) Prepare samples:

[0093] The average individual weight of thick-shelled mussels is 60.15g, the average width of the shell under healthy conditions is 4.5cm, and the average shell height is 9.1cm. It comes from the Dongji Island Aquaculture Base (Zhoushan City, Zhejiang Province, China), and the filtered seawater is kept during the breeding period. Continue to oxygenate and cultivate at a temperature of 24-25°C and a pH value of 8.0.

[0094] (2) LPS and Cu 2+ challenge:

[0095] 1) LPS challenge: For LPS challenge, 80 adult thick-shelled mussels were randomly divided into two groups, one group of 40 individuals (control group) was injected with PBS (pH7.4) for adductor muscles and the other group of 40 individuals (Challenge group) to conduct LPS challenge. Challenged mussel...

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