A dominant negative effect mutant of heat shock transcription factor 1 and its application

A transcription factor, heat shock technology, applied in application, genetic engineering, plant genetic improvement and other directions, to avoid human toxicity and side effects, grow better, and avoid environmental pollution.

Active Publication Date: 2021-07-27
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no application of fungal Hsf1 dominant-negative mutants to control fungal contamination.

Method used

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  • A dominant negative effect mutant of heat shock transcription factor 1 and its application
  • A dominant negative effect mutant of heat shock transcription factor 1 and its application
  • A dominant negative effect mutant of heat shock transcription factor 1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, construction of dn-Hsf1 expression vector

[0062] Studies in mammals have shown that deletion of the catalytic domain of about 150 amino acid residues at the C-terminus of the heat shock transcription factor 1 HSF1 protein can generate a mutant dn-cHSF1 with a dominant negative effect, which can inhibit the normal HSF1 functions in animals (DOI: 10.1021 / acschembio.5b00740). Therefore, the present inventors constructed a fungal-derived Hsf1 dominant-negative mutant dn-Hsf1 to inhibit the function of normal Hsf1 in fungi, thereby inhibiting the growth of fungi and achieving the purpose of preventing and controlling fungal contamination.

[0063] The present invention uses the amino acid sequence of the human heat shock transcription factor 1 HSF1 protein to find the predicted homologous protein Hsf1 (No. XP_002374014) and its coding gene (No. AFLA_025030) in Aspergillus flavus through the NCBI BLASTP homologous alignment method. The amino acid sequence sim...

Embodiment 2

[0069] Embodiment 2, expression vector pPTR I -dn-hsf1 Aspergillus flavus

[0070] The expression vector plasmid pPTR I -dn-hsf1 The protoplasts of Aspergillus flavus WT strain were introduced, and the transformants of Aspergillus flavus were screened on the recovery medium containing pyrithiamine. After the transformation plate was cultured at 37°C for 5 days, the results were as follows: figure 2 As shown: the first and second culture plates are protoplasts without transformed nucleic acid. Since the first culture plate does not contain pyrithiamine screening pressure, many colonies grow on the culture plate to form a lawn, indicating that it is used for transformation The amount of protoplasts was sufficient, but the second culture plate was added with pyridinethiamine, and no clones were grown, indicating that the screening pressure of pyrithione was effective. The third culture plate was the protoplast transformed with pPTR I empty vector, and 10 clones grew out und...

Embodiment 3

[0071] Example 3, Xylose promoter induced expression dn-Hsf1 inhibits the growth of Aspergillus flavus

[0072] In order to confirm the inhibitory effect of dn-Hsf1 on the growth of Aspergillus flavus, the present invention further expresses dn-Hsf1 protein from Aspergillus flavus hsf1 gene promoter P hsf1 Control changed to use a xylose-inducible promoter P xy1P to control (build strategies such as image 3 Shown in A), constructed by fusion PCR method containing upstream homologous sequences up , derived from Aspergillus fumigatus pyrG Nutritional screening marker gene, derived from Penicillium chrysogenum P xylP Xylose-inducible expression promoter, coding nucleotide and terminator of dn-Hsf1 T hsf1 The fusion fragment of P xylP -dn-hsf1-T hsf1 Fragment, transformed and integrated into the Aspergillus flavus genome, to obtain the inducible P xylP -dn- hsf1 strain. Observe the effects of dn-Hsf1 non-expression and induced expression on the growth of ...

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Abstract

The invention discloses a heat shock transcription factor 1 dominant negative effect mutant dn-Hsf1 and its application, belonging to the field of biotechnology. Human and A. flavus ( Aspergillus flavus ) in the heat shock transcription factor 1 HSF1 protein, and a total of 213 amino acid residues from the 576th to the 788th position of the C-terminal of the Aspergillus flavus Hsf1 protein were deleted to obtain the heat shock transcription factor 1 dominant negative mutant dn in Aspergillus flavus The amino acid sequence of the -Hsf1 protein is shown in SEQ ID NO.1, and the coding nucleotide sequence is shown in SEQ ID NO.2. The dominant-negative mutant dn-Hsf1 can inhibit the function of normal Hsf1 in fungi, thereby inhibiting the growth of fungi, and is used in the prevention and control of fungal pollution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a heat shock transcription factor 1 dominant-negative effect mutant and application thereof. Background technique [0002] Fungi are a class of eukaryotic organisms widely distributed in nature, which are both beneficial and harmful to human life. Many fungi provide us with food sources. As important species in the fermentation industry and food processing industry, a variety of secondary metabolites produced by them have important application value in industrial production and medicine. There are also many fungi that cause great harm to human health and socio-economics. Many fungi, such as Aspergillus fumigatus ( Aspergillus fumigatus ) and Candida albicans ( Cadida albicans ), can infect the human body, causing local tissue and organ or systemic fungal infection and even death. Many fungi can also parasitize in animals, plants and their processed products, and pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/38C12N15/31C12N15/80C12N15/62C12N15/65C12N1/19C12R1/67
CPCC07K14/38C07K2319/00C12N15/65C12N15/80
Inventor 聂鑫怡王银春李博文张轶薛杨王佳琪张双焘汪世华
Owner FUJIAN AGRI & FORESTRY UNIV
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