Application of thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring

A metallothionein, thick-shelled mussel technology, applied in the field of biological sciences, can solve the problems of sea area destruction, seawater quality, deterioration of environmental conditions, pollution, etc., and achieve a highly specific effect

Inactive Publication Date: 2018-12-21
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the rapid development of coastal cities' industrialization, the marine environment has been further damaged, which has brought great damage to the sea area where thick-shelled mussels live and reproduce. The water quality and environmental conditions of seawater have gradually deteriorated and the pollution is serious

Method used

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  • Application of thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring
  • Application of thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring
  • Application of thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The metallothionein gene of thick shell mussel is obtained by using gene cloning technology, the nucleotide sequence of the gene is SEQ ID NO.7, and the full length of the sequence is 314bp. Metallothionein MT is a low-molecular-weight, cysteine-rich, thermostable, inducible non-enzymatic protein that is ubiquitous in organisms. It can stably regulate intracellular metal levels and participate in the regulation of essential metal elements in organisms. and detoxification of non-essential metals.

[0037] Above-mentioned gene cloning comprises the steps:

[0038] 1) Extraction of total RNA:

[0039] a) Take a clean 1.5ml centrifuge tube (RNAase free), add 500μl Trizol (4°C) with an RNAase-free pipette tip, use DEPC-treated tweezers to pick up about 30mg of liver tissue sample and immerse it in Trizol, and use an electric homogenizer to homogenize 30s, add 500μl Trizol;

[0040] b) Gently shake the centrifuge tube to fully dissolve the tissue into Trizol, and let stand...

Embodiment 2

[0085] The metallothionein gene of thick shell mussel was obtained by gene cloning technology, and the further optimization scheme is as follows:

[0086] Total RNA was extracted using Trizol Regent, guanidine phosphate and thiourea. Trizol Regent contained 0.03% guanidine phosphate and 0.01% thiourea. Trizol Regent, guanidine phosphate, and thiourea can play a beneficial role. On the one hand, they can quickly penetrate into tissues, inactivate endogenous RNases, do not cause RNA degradation, facilitate the subsequent extraction of RNA, and have strong cleavage ability. Lying cells and tissue samples in a short period of time can rapidly denature the proteins released by the cells and inhibit the RNase activity released by the cells, effectively inhibiting the degradation of RNA in the sample and maintaining the integrity of the RNA; on the other hand, assisting in the denaturation of proteins Dissolve the substances such as chloroform in the subsequent addition, so that the ...

Embodiment 3

[0088] Tissue-specific expression analysis of MT gene in Mussel sclerosus

[0089] The specific steps are:

[0090] 1) Total RNA extraction from different tissues:

[0091]Randomly select 3 thick-shelled mussels, extract their liver, mantle, serum, adductor muscle, gills, and gonads, and extract RNA to prepare for tissue-specific experiments on thick-shelled mussels. According to the Trizol of TaKaLa Company The method recommended by the Total RNA extraction kit was carried out. The obtained total RNA was detected by electrophoresis on a 1.5% non-deformed agarose gel, and its concentration and A260 / A280 value were detected by a UV spectrophotometer (Bio-Rad, USA);

[0092] 2) cDNA template synthesis

[0093] M-MLV (RNAase H - ) Reverse transcription kit, reverse-transcribe the total RNA collected from each tissue into cDNA, reverse 3 copies at a time, store it in a -80°C ultra-low temperature refrigerator after completion, and then use the NANODROP 2000 nucleic acid protein...

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Abstract

The invention discloses application of a thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring. The thick shell mussel metallothionein gene is obtained through adopting a gene cloning technology; the total length of a gene cDNA sequence is 314bp; according to the application of the thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring, the concrete monitoring method is characterized by extracting RNA in a thick shell mussel liver subjected to heavy metal pollution, synthesizing the cDNA; using a fluorescent quantitation technology for detecting the relative transcript level of a metallothionein enzyme in the liver. The application of the thick shell mussel metallothionein gene in ocean heavy metal pollution monitoring has the beneficial effects that the situation of the thick shell mussel metallothionein gene subjected to heavy metal explosion and the heavy metal pollution pressure can be predicated from the molecular level, the specificity is high, the expression quantity of an MT gene and the heavy metal concentration in an organism have a remarkable positive correlation property, and the guidance is provided for an aquaticwater environmental pollution early warning mark.

Description

technical field [0001] The invention relates to the technical field of biological sciences, in particular to the application of the mussel metallothionein gene in the monitoring of marine heavy metal pollution. technical background [0002] thick-shelled mussels ( Mytilus coruscus ) is a common economical bivalve mollusc belonging to the genus Mytilus of the family Mytilidae in the order Mytiloida. There are traces of distribution in all regions, and the resources in the coast of Zhejiang are the most abundant. However, with the rapid development of coastal cities' industrialization, the marine environment has been further damaged, which has brought great damage to the sea area where the mussels live and reproduce. The water quality and environmental conditions of the seawater have gradually deteriorated and the pollution is serious. Bivalve molluscs in the mollusk have a strong bioaccumulation effect on heavy metals in the marine environment. They are often used to monito...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C07K14/825C12Q1/6876
CPCC07K14/825C12N15/10C12Q1/6876C12Q2600/158
Inventor 刘慧慧葛德隆迟长凤黄伟
Owner ZHEJIANG OCEAN UNIV
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