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Barbus tetrazona heat shock protein PtHsp70 and application thereof

A heat shock protein, tiger skin technology, applied in the field of genetic engineering, can solve the problems of sequence structure and expression mechanism that have not been reported yet

Pending Publication Date: 2019-01-18
BEIJING FISHERIES RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the cDNA sequence of hsp70 has been amplified from a variety of carps. Some studies have reported the expression of heat shock proteins in carps under temperature stimulation, but the sequence structure and expression mechanism of hsp70 gene in tiger fish Not yet reported

Method used

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  • Barbus tetrazona heat shock protein PtHsp70 and application thereof
  • Barbus tetrazona heat shock protein PtHsp70 and application thereof
  • Barbus tetrazona heat shock protein PtHsp70 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 PtHSP70 gene cloning

[0018] Three 8-month-old tiger skin fishes cultured in our laboratory were taken, anesthetized with MS-222, dissected, and the brain, gills, liver and muscle tissues were collected respectively, and the same kinds of tissues were mixed and stored, and the total RNA was immediately extracted with TRizol reagent. The Nanodrop2000 Nucleic Acid Protein Concentration Detector was used to measure the total RNA concentration and contamination, the RNA integrity was detected by agarose gel electrophoresis, and stored at -80°C for later use.

[0019] Using the total RNA of each tissue as a template, the first strand of cDNA was synthesized using a reverse transcription kit (TaKaRa, 6110A) according to the operating method and reagent instructions. Chain polymerase reaction (PCR) was performed using the first strand of cDNA as a template, in which primer F1: 5'-TCGAAGCGAGCTCTGGTGATGGACGTGT-3', R1: 5'-TCACCAACGACAAGGGCAGGCTGAGCAA-3'. PCR reaction ...

Embodiment 2

[0021] Example 2 Analysis of HSP70 gene expression level in tiger fish and zebrafish under low temperature stress

[0022] Tiger fish and zebrafish belong to the Cyprinidae family and are native to freshwater basins in Southeast Asia, and have a high degree of evolutionary similarity. Thirty 8-month-old adult tiger fish and zebrafish were randomly divided into experimental group and control group, with 15 fish in each group. Each group was divided into 3 tanks with 5 fish in each tank. Adopt the method of gradient cooling, cool down the tiger skin fish cultured at 27℃ by 2℃ every 24hr, when the temperature reaches 19℃, continue to culture at this temperature for 24hr, dissect muscle tissue after MS-222 anesthesia, and extract total RNA with TRizol reagent , using a reverse transcription kit (TaKaRa, 6110A) to synthesize the first strand of cDNA and use it as a template for qPCR experiments. Real-time fluorescent quantitative PCR (qPCR) technology was used to detect the mRNA e...

Embodiment 3

[0027] Example 3 Preparation of linearized vector and target fragment

[0028] Carry out Bgl II and Sal I double enzyme digestion on the commercialized plasmid pAcGFP1-C1. The enzyme digestion system is: 2 μL 10×Buffer O, 600 ng of pAcGFP1-C1 plasmid, 1 μL each of endonuclease Sal I and Bgl II, deionized water Bring the volume up to 20 μL.

[0029] Primers were designed according to the gene sequence information of the target fragment, and restriction sites agatct (Bgl II) and gtcgac (Sal I) were introduced into the upstream and downstream of the fragment respectively, wherein the sequence of primer F2 was: 5'-agatctATGTCATCTGCAAAAGGAGTTGCTATTG-3'; the sequence of primer R2 was : 5'-gtcgacTTAATCCACCTCCTCGATAGTGGGC-3', prepare the solution according to the following reaction system: 0.25μL Takara Ex Taq, 5μL 10×PCR buffer (Mg 2+ free), 4 μL MgCl 2 , 4μL dNTPmixture (0.25mM each), 0.4μL (<500ng) cDNA template, 2μL each of primer F (10μM) and primer R (10μM), make up the volume...

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Abstract

The invention discloses barbus tetrazona heat shock protein PtHsp70 and application thereof. The nucleotide sequence of the PtHsp70 gene is shown in a sequence table SEQ ID NO: 1. The complete codingsequence of barbus tetrazona PtHsp70 cDNA is cloned; the specific expression of PtHsp70 in different tissues is detected and analyzed by a qRT-PCR technology; PtHsp70 is found to be expressed in a large amount under a low temperature condition, and can resist the harm of low temperature to a barbus tetrazona cub, and the protein is also expressed in a large amount under the condition of water bodyeutrophication, and can be used as a dual molecular marker for water body low-temperature stress and water body eutrophication.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tiger skin fish heat shock protein PtHsp70 and an application thereof. Background technique [0002] Fish is a temperature-changing animal, which is very susceptible to environmental influences such as temperature, osmotic pressure, and dissolved oxygen, and produces heat shock protein (heat shocking protein, HSP) at the cellular level. HSP is a type of highly conserved protein among different life forms. Protein, which can resist environmental stress and promote cell growth. Heat shock protein 70kDa (HSP70) can quickly and accurately respond to temperature stimulation, heavy metal stress, free radical attack and microbial infection, so it has been deeply studied as a biomarker. HSP70 is mainly involved in protein folding and transport in the form of intracellular molecular chaperones, and plays a key role in cell growth, survival and apoptosis. In aqu...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12C12N15/63C12N15/11C12Q1/6888
CPCC07K14/461C12Q1/6888C12Q2600/124C12Q2600/158
Inventor 刘丽丽朱华张蓉朱建亚王晓雯
Owner BEIJING FISHERIES RES INST
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