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A method for prokaryotic expression of Liriomyza trifolii heat shock transcription factor protein

A technology of L. triloba, prokaryotic expression, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems such as research gaps in the regulation mechanism of heat shock proteins, and achieve the effect of improving protein expression.

Active Publication Date: 2022-04-26
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been some studies on the expression of heat shock proteins in Liriomyza trifoliata, the research on the regulation mechanism of these heat shock proteins is still blank.

Method used

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  • A method for prokaryotic expression of Liriomyza trifolii heat shock transcription factor protein
  • A method for prokaryotic expression of Liriomyza trifolii heat shock transcription factor protein

Examples

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Effect test

Embodiment 1

[0025] Example 1 Different concentrations of IPTG induce the expression of Hsf1

[0026] A method for prokaryotic expression of Liriomyza trifolii heat shock transcription factor protein according to the present invention, the specific steps are as follows:

[0027] (1) Analyze the open reading frame (ORF) of the Hsf gene of Liriomyza trifoliata obtained from the transcriptome of high and low temperature treatment of Liriomyza trifoliata (PRJNA575513), and design with specific restriction sites, BamHI and XhoI The specific primers are used to amplify the target fragment, and the Hsf gene sequence is SEQ ID NO.1.

[0028] (2) Recover and clone the PCR product and extract the plasmid after the sequencing is correct.

[0029] (3) Construction of prokaryotic expression vector Restriction restriction. The positive clone recombinant plasmid and the prokaryotic expression vector plasmid (pET28a) carrying the target gene were subjected to double enzyme digestion respectively. Enzym...

Embodiment 2

[0035] A method for prokaryotic expression of Liriomyza trifolii heat shock transcription factor protein according to the present invention, the specific steps are as follows:

[0036] (1) Analyze the open reading frame (ORF) of the Hsf gene of Liriomyza trifoliata obtained from the transcriptome of high and low temperature treatment of Liriomyza trifoliata (PRJNA575513), and design with specific restriction sites, BamHI and XhoI The specific primers are used to amplify the target fragment, and the Hsf gene sequence is SEQ ID NO.1.

[0037] (2) Recover and clone the PCR product and extract the plasmid after the sequencing is correct.

[0038] (3) Construction of prokaryotic expression vector Restriction restriction. The positive clone recombinant plasmid and the prokaryotic expression vector plasmid (pET28a) carrying the target gene were subjected to double enzyme digestion respectively. Enzyme digested in water bath at 37°C for 1 hour, run the gel for gel recovery, and use ...

Embodiment 3

[0044] A method for prokaryotic expression of Liriomyza trifolii heat shock transcription factor protein according to the present invention, the specific steps are as follows:

[0045] (1) Analyze the obtained open reading frame (ORF) of the Liriomyza trifolia Hsf gene, design specific primers with specific restriction sites (BamHI and XhoI), and amplify the target fragment.

[0046] (2) Recover and clone the PCR product and extract the plasmid after the sequencing is correct.

[0047] (3) Construction of prokaryotic expression vector Restriction restriction. The positive clone recombinant plasmid and the prokaryotic expression vector plasmid (pET28a) carrying the target gene were subjected to double enzyme digestion respectively. Enzyme digested in water bath at 37°C for 1 hour, run the gel for gel recovery, and use T4 DNA ligase for ligation. Mix well by whipping, centrifuge briefly, and incubate overnight at 16°C. After ligation, the recombinant plasmid was transformed i...

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Abstract

The invention discloses a method for prokaryotic expression of Liriomyza trifolia heat shock transcription factor protein, which includes two parts: construction of protein expression vector and induced expression of prokaryotic expression vector, specifically: design with a specific enzyme cutting site (BamHI and XhoI) specific primers to amplify the target fragment. The PCR products were recovered, cloned, and plasmids were extracted after the sequencing was correct. Construction of prokaryotic expression vector Restriction restriction. Preparation of expression bacteria. The optimal conditions for inducing expression were explored. The expression form of the target protein and identification by Western blot. This experiment not only explored the optimal IPTG concentration to increase the prokaryotic expression level of the heat shock transcription factor protein of Liriomyza trifoliata, improved the efficiency and effect of the experiment, but also provided a comprehensive study on the transcriptional regulation mechanism of the entire Hsp family gene, and its role in The role of Liriomyza sativae in temperature tolerance and invasion transmission has important implications.

Description

technical field [0001] The invention relates to biotechnology, in particular to a prokaryotic expression method for heat shock transcription factor protein of Liriomyza trifolii. Background technique [0002] There are many species of Liriomyza sativae, and the interspecific competition and substitution are rapid. Liriomyza trifolii, Liriomyza trifolii, L. sativae and L. huidobrensis are three polyphagous liriomyza that are seriously harmful. Although the three closely related species share similar morphological characteristics and overlapping ecological niches, Liriomyza trifolii has stronger invasion and competition abilities than its relatives. Temperature is an important factor affecting the interspecific competition and replacement of invasive Liriomyza sativae. During the process of invasion and spread, Liriomyza sativus developed corresponding tolerance against extreme temperature to adapt to environmental changes, but there are few studies on its internal molecular...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N15/66
CPCC07K14/43577C12N15/70C12N15/66
Inventor 杜予州王禹程常亚文
Owner YANGZHOU UNIV
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