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Cloning method of perinereis aibuhitensis metal binding protein gene

A metal-binding protein and a technology for B. biloba, applied in the field of genetic engineering, can solve the problems of inability to deeply study functional characteristics, regulatory mechanisms, affecting the wide application of B. biloba, etc., and achieve the effect of being beneficial to cost and rich in sources.

Inactive Publication Date: 2012-01-04
DALIAN OCEAN UNIV
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Problems solved by technology

[0023] So far, the research on the metal-binding protein (MPII) of P. didentate is mainly focused on the detection and analysis of its protein level, and there is no report of cloning the gene, so that it is impossible to study its functional characteristics and regulation mechanism in depth. Affected the wide application of P. didentate in environmental protection

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  • Cloning method of perinereis aibuhitensis metal binding protein gene
  • Cloning method of perinereis aibuhitensis metal binding protein gene
  • Cloning method of perinereis aibuhitensis metal binding protein gene

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Embodiment 1

[0043] The method for cloning the metal-binding protein gene of P. didentate is as follows:

[0044] one. Extract and purify the total RNA of P. didentate, and synthesize the first strand of cDNA;

[0045] 1. Extraction and purification of total RNA: Trizol from Invitrogen was used. The electrophoresis of the extracted and purified total RNA is as follows: figure 1 shown. Wherein, M is the molecular weight marker, and 1-2 is the purified total RNA.

[0046] 2. cDNA first-strand synthesis: using the Takara RNA PCR Kit (AMV) Ver.3.0 kit, mix the following reagents:

[0047] Mgcl 2 2μl

[0048] 10× reverse transcription buffer 1 μl

[0049] RNase Free dH 2 O 3.75 μl

[0050] dNTP 1 μl

[0051] RNase inhibitor 0.25 μl

[0052] AMV reverse transcriptase 0.5 μl

[0053] Adapter primer Oligo dT 0.5 μl

[0054] Total RNA 1 μl

[0055] 10 μl total volume

[0056] Reverse transcription reaction conditions: 30°C...

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Abstract

The invention discloses a cloning method of a perinereis aibuhitensis metal binding protein gene. According to the invention, a specific segment amplification method and a RACE amplification method are designed, and a perinereis aibuhitensis metal binding protein gene is cloned from perinereis aibuhitensis body wall muscle total RNA. With the method, the MP II gene expression regularity of perinereis aibuhitensis suffered from stresses of pollutants such as heavy metals can be detected with a real-time fluorescence quantitative PCR technology. Therefore, the method provides a basis for the study of the mechanism of heavy metal regulation on perinereis aibuhitensis MP II gene. Also, the method assists in establishing a basis for screening molecular biomarkers for marine sedimentary environment pollution early-stage ecological risk forecasting. With the method, perinereis aibuhitensis with a low cost and an abundant source can be widely applied in environment protection.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for cloning the metal-binding protein (MPII) gene of the clam worm. Background technique [0002] Polychaetes belong to the phylum Annelida, and are dominant benthic animals widely distributed in the coastal and estuary tidal flats of the ocean, and are also an important link in the energy flow and material cycle of marine ecosystems. Because of its large size, long life cycle, slow action, wide distribution, and strong tolerance and bioavailability to many persistent pollutants, it has been used as an indicator organism for environmental pollution assessment of marine sediments and a tool for toxicology research. model animals. For example, Bryan et al. found that the variegated clamworm ( Nereis diversicolor ) exposed to Cd 2+ 192?h LC 50 It reaches 100?mg / L, far exceeding the tolerance range of general marine invertebrates (0.1-10?mg / L). Studies hav...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10
Inventor 周一兵杨大佐何洁王斌陈雪赵欢
Owner DALIAN OCEAN UNIV
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