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Bay scallop heat shock protein 70 gene promoter and application thereof

A heat shock protein and promoter technology, applied in the fields of molecular biology and genetic engineering, can solve the problem of no heat-inducible promoter, etc., and achieve the effect of strong promoter activity

Active Publication Date: 2013-07-10
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In aquatic animals, no heat-inducible promoters have been studied

Method used

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  • Bay scallop heat shock protein 70 gene promoter and application thereof
  • Bay scallop heat shock protein 70 gene promoter and application thereof
  • Bay scallop heat shock protein 70 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 RT-PCR detection of heat-stressed scallop heat shock protein 70mRNA expression

[0031] Bay scallops for experiments were purchased from Qingdao Aquatic Products Market, and kept in seawater at (17±1)°C for a week, and then subjected to continuous high temperature stimulation experiments. 5 scallops were taken as the blank group, while the other 25 scallops were placed in seawater at 28°C for heat shock as the treatment group. At 1h, 2h, 4h, 8h and 12h after stimulation, the gills of scallops were frozen and stored at -80°C for RNA extraction. Using primer oligo dT (18) and reverse transcriptase M-MLV at 42°C for 1h. With the scallop housekeeping gene β-actin as an internal reference, the mRNA expression of the AiHSP70 gene was detected by RT-PCR (see figure 1 ).

[0032] The result is as figure 1 As shown, after heat shock at 28°C for 1 hour, the expression level of heat shock protein 70 mRNA in bay scallops rose rapidly and reached the maximum value, wh...

Embodiment 2

[0033] Example 2 Cloning of the promoter of the heat shock protein 70 gene of bay scallop

[0034] 1. Using the restriction enzymes (Dra I, EcoR V, PvμII & StμI) of the GenomeWalkerUniversal Kit (Catalog No. 638904) of Clontech Company (www.Clontech.com) to construct four DNA endonuclease libraries.

[0035] 2. Use two rounds of two-step PCR method to clone the 5'-end sequence, the specific steps are as follows:

[0036](1) The total system for the first round of PCR is 50 μl, including 38.5 μl sterile double distilled water, 5 μl 10×PCR buffer, 3 μl dNTP (10 mM), 1 μl linker primer (AP1: 5'-GTAATACGACTCACTATAGGGC-3', 10 μM), 1 μl gene Specific primer (SEQ ID NO.2, 10 μM)), 0.5 μl rTaq polymerase (TaKaRa) and 1 μl restriction library DNA constructed as described in Step 1 were used as templates. The PCR program is: 7 cycles: 94°C for 25s, 72°C for 3min; 32 cycles: 94°C for 25s, 67°C for 3min; 1 cycle: 67°C for 7min.

[0037] (2) The second round of two-step PCR uses the PCR ...

Embodiment 3

[0085] Example 3 Bay Scallop Heat Shock Protein 70 Gene Promoter (pAiHSP70) Activity Detection

[0086] 1. Construction of pGL-3-Basic-pAiHSP70 recombinant vector: 1) Synthesis of primers SEQ ID NO.4 (5'-GGTACCATAACGGCTTGCCATACTAAC-3') and SEQ ID NO.4 (5'-GGTACCATAACGGCTTGCCATACTAAC-3') and SEQ ID NO. ID NO.5 (5'-AGATCTTTGCTAAAAACAAAAACGAAAT-3'), was amplified by PCR with Takara high-fidelity Taq enzyme. The reaction system with a total volume of 50 μl contains 38.5 μl sterile double distilled water, 5 μl 10×PCR buffer, 3 μl dNTP (10 mM), 1 μl primer SEQ ID NO.4 (10 μM), 1 μl SEQ ID NO.5 (10 μM) ), 0.5 μl high-fidelity Taq polymerase (TaKaRa) and 1 μl whole genome DNA as template (50ng / ul). The PCR program is: 1 cycle: 94°C for 5 min; 32 cycles: 94°C for 30 s, 55°C for 30 s, 72°C for 90 s; 1 cycle: 72°C for 10 min. PCR products were purified using a gel extraction kit (TaKaRa).

[0087]2) The recovered PCR product and the empty promoter-free vector pGL-3-Basic (Promega) wer...

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Abstract

The invention belongs to the technical fields of molecular biology and gene engineering, and relates to a sequence of a heat shock promoter, namely a bay scallop heat shock protein 70 gene promoter, a cloning method for the bay scallop heat shock protein 70 gene promoter, and construction of a recombinant vector comprising the promoter, wherein the recombinant vector can be used for expressing exogenous genes in host cells. The invention provides a bay scallop heat shock protein 70 gene promoter and a cloning method thereof, a recombinant vector comprising the bay scallop heat shock protein 70 gene promoter, and a recombinant host cell. By a molecular biology method, the expression modes of bay scallop heat shock protein 70 gene mRNA and reporter gene Luciferase under the control of the segment are analyzed, and results show that the promoter is subjected to heat stress for inducing and controlling and the exogenous genes can be promoted to be highly expressed in Hela cells.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a promoter of heat shock protein 70 gene of bay scallop and application thereof. Background technique [0002] In recent years, my country's shallow seashell culture has made great progress, but with climate warming, high temperature in summer has become the main environmental factor restricting the development of aquaculture industry. When organisms are subjected to heat shock stress (a temperature 5°C higher than the normal growth temperature), they can rapidly induce the synthesis of heat shock proteins, which were first discovered by Ritossa in Drosophila in 1962. In addition to temperature, many damage factors and stress stimuli (such as hypoxia, heavy metal ions, virus infection, free radicals, etc.) can induce heat shock responses, and the synthesized heat shock proteins (HSPs) play a role in the reconstruction of cellular homeostasis. important r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
Inventor 宋林生杨传燕王玲玲邱丽梅张峘
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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