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Oyster cell apoptosis gene SMAC gene and application thereof in preparation of pathology detecting and diagnostic reagent

An oyster and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of lack of disease detection methods, production fluctuations, and high risks of oyster farming.

Active Publication Date: 2018-09-28
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of marine shellfish in China is mainly based on traditional farming, which has a large farming area and high energy consumption. The output fluctuates due to changes in various conditions. Moreover, due to the lack of effective disease detection methods, with the expansion of the farming scale, the oyster Breeding risks are also increasing

Method used

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  • Oyster cell apoptosis gene SMAC gene and application thereof in preparation of pathology detecting and diagnostic reagent
  • Oyster cell apoptosis gene SMAC gene and application thereof in preparation of pathology detecting and diagnostic reagent
  • Oyster cell apoptosis gene SMAC gene and application thereof in preparation of pathology detecting and diagnostic reagent

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Embodiment 1

[0032] 1. RNA extraction and reverse transcription

[0033] a). After adding Trizol to Hong Kong giant oyster cells or tissues, grind them with a tissue grinder, and place them at room temperature for 5 minutes to fully lyse them.

[0034] b). Centrifuge at 12,000rpm for 5min and discard the precipitate.

[0035] c). Add chloroform to 200ul chloroform / ml Trizol, vortex and mix well, and place at room temperature for 15min.

[0036] d). Centrifuge at 12,000 g for 15 min at 4°C.

[0037] e). Aspirate the upper aqueous phase into another centrifuge tube.

[0038] f). According to 0.5ml isopropanol / ml Trizol, add isopropanol and mix well, and place at room temperature for 5-10min.

[0039] g). Centrifuge at 12,000g at 4°C for 10 minutes, discard the supernatant, and sink the RNA to the bottom of the tube.

[0040] h). Add 75% ethanol to 1ml 75% ethanol / ml Trizol, shake the centrifuge tube gently, and suspend the precipitate.

[0041] i). Centrifuge at 8,000g at 4°C for 5 minu...

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Abstract

The invention discloses an oyster cell apoptosis gene SMAC gene and an application thereof in the preparation of a pathology detecting and diagnostic reagent. The invention discovers the symbolic geneSMAC gene of the cell apoptosis of the species, and the nuclear antigen sequence is shown as from the 50th bp to the 1006th bp of the SEQ ID NO.1. The amino acid sequence (the amino acid sequence isshown in the SEQ ID NO. 2) of the gene coding has high species specificity, the similarity of the amino acid sequence in ostrea is 60%-88%, the evolution relationship is close, and the similarity withthe homologous proteins of other species is 30% or below, so that the evolution relationship is far. The quantitative polymerase chain reaction (PCR) experiment shows that the SMAC gene is up-regulated after the infection of vibrio alginolyticus and staphylococcus haemolyticus, can be used as a standard for detecting the apoptosis of the oyster cells, provides a positive and accurate warning signal for the prevention of oyster diseases, and prevents the expansion of the disaster.

Description

Technical field: [0001] The present invention relates to the molecular mechanism of shellfish immunity and cell apoptosis, and more specifically relates to a SMAC (secondmitochondria-derived activator of caspases) screened from Hong Kong giant oyster (Crassostrea hongkongensis) blood cell subtractive hybridization library stimulated by Vibrio The nucleotide sequence and amino acid sequence of the gene, its preparation method and the application of the gene. Background technique: [0002] my country is the largest oyster farming country in the world, and according to statistics, its annual output accounts for more than 80% of the world's total output. According to the 2015 China Fishery Statistical Yearbook, the oyster farming area reached 141,500 hectares in that year, and the output was about 4.5734 million tons. Therefore, oyster farming occupies a pivotal position in my country's coastal economic and social development and is an important pillar industry; The Hong Kong gi...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12Q1/6883C12N15/11
CPCC07K14/43504C12Q1/6883C12Q2600/158C12Q2600/166Y02A50/30
Inventor 向志明秦艳平马海涛李军张扬肖述张跃环喻子牛
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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