Cloning Method of Cytochrome Oxidase Gene of Peri. didentatus

A technology of Nereis didentate and cytochrome, which is applied in the field of genetic engineering, can solve the problems that affect the wide application of Nereis didentate and the inability to study the gene of cytochrome oxidase in Nereis didentate, and achieve a cost-effective Effect

Inactive Publication Date: 2011-12-21
DALIAN OCEAN UNIV
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Problems solved by technology

However, so far, there has been no report on the cloning of the C. didentatum cytochrome oxidase gene, so that it is impossible to study the molecular level transcription mechanism

Method used

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  • Cloning Method of Cytochrome Oxidase Gene of Peri. didentatus
  • Cloning Method of Cytochrome Oxidase Gene of Peri. didentatus
  • Cloning Method of Cytochrome Oxidase Gene of Peri. didentatus

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Embodiment 1

[0028] The cloning method of the cytochrome oxidase gene of the bidentate silkworm is as follows:

[0029] One. Extract and purify the total RNA of the bidentate silkworm, synthesize the first strand of cDNA;

[0030] 1. Extraction and purification of total RNA: Use RNAiso Pius from Bao Bioengineering (Dalian) Co., Ltd. The extracted and purified total RNA electropherogram is as follows figure 1 Shown. Among them, M is a molecular weight marker, 1, 2, and 3 are purified total RNA.

[0031] 2. Synthesis of the first strand of cDNA: Use the RNA PCR Kit (AMV) Ver.3.0 kit of Bao Biological Company to mix the following reagents:

[0032] Mgcl 2 2 μl

[0033] 10×Reverse transcription buffer 1 μl

[0034] Rnase Free dH 2 O 3.75 μl

[0035] dNTP 1 μl

[0036] RNase inhibitor 0.25 μl

[0037] AMV reverse transcriptase 0.5 μl

[0038] Adapter primer Oligo dT 0.5 μl

[0039] Total RNA 1 μl

[0040] Total volume 10 μl

[0041] Reverse transcription reaction...

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Abstract

The invention discloses a method for cloning cytochrome oxidase gene of Nereis bidentata, which is to clone the total RNA of Nereis bidentata body wall muscle by designing specific fragment amplification and RACE amplification. The cytochrome oxidase gene of Nereis dentata can be detected by real-time fluorescent quantitative PCR technology under the stress of persistent organic pollutants. The mechanism of oxidase gene expression and regulation provides a basis, and also lays the foundation for screening molecular biomarkers for early ecological risk prediction of marine sediment environmental pollution, which is conducive to the wide application of the low-cost and abundant sources of the clamworm in environmental protection.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for cloning the cytochrome oxidase gene (CYP4) of the bidentate silkworm. Background technique [0002] Polychaete belongs to the phylum Annelid, which is a dominant benthic animal widely distributed in the ocean nearshore and estuarine tidal flats. It is also an important link in the energy flow and material circulation of the marine ecosystem. Because of its large size, long life cycle, slow action, wide distribution, and strong tolerance and bioavailability to many persistent pollutants, it has always been used as an indicator organism for marine sediment environmental pollution evaluation and as an indicator of toxicological research. Model animals. For example, the living environment of silkworm populations is often polluted by petroleum hydrocarbons. Some silkworm populations show high tolerance to petroleum hydrocarbons (PHs) and have certain degradation...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/10
Inventor 周一兵杨大佐何洁王斌陈雪赵欢
Owner DALIAN OCEAN UNIV
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