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Transcriptional regulation factor derived from trichoderma reesei and coding gene and application of transcriptional regulation factor

A transcriptional regulator, Trichoderma reesei technology, applied in applications, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2013-04-24
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no engineered strain modified by molecular biology for large-scale industrial production

Method used

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  • Transcriptional regulation factor derived from trichoderma reesei and coding gene and application of transcriptional regulation factor
  • Transcriptional regulation factor derived from trichoderma reesei and coding gene and application of transcriptional regulation factor
  • Transcriptional regulation factor derived from trichoderma reesei and coding gene and application of transcriptional regulation factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Full gene cloning of the transcriptional regulator TrPac1 in Trichoderma reesei

[0049] 1. Cloning of TrPac1 gene DNA sequence

[0050] Genomic DNA was extracted from Trichoderma reesei strain QM9414 (preserved in the inventor's laboratory) as a template, and then primers were designed according to the information in the Trichoderma reesei genome database, and the target fragment was obtained by high-fidelity PCR.

[0051] (1) Extraction of Trichoderma reesei genomic DNA

[0052] Wash the Trichoderma reesei spores growing on the PDA plate with sterile water, and filter with sterile three-layer lens cleaning paper after washing, and dilute to 10 6 Pieces / ml; 1ml of spore suspension was inoculated into an Erlenmeyer flask containing 80ml of CM medium, and cultured at 28°C and 180rpm for 24h. The cultured hyphae were filtered with sterilized three-layer lens cleaning paper, and the hyphae were washed 2-3 times with distilled water, then collected, blotted dry with abso...

Embodiment 2

[0062] Example 2: Cloning of the cDNA sequence of the transcriptional regulator TrPac1 gene from Trichoderma reesei

[0063] 1. Extraction of total RNA from Trichoderma reesei

[0064] Wash the Trichoderma reesei spores growing on the PDA plate with sterile water, and filter with sterile three-layer lens cleaning paper after washing, and dilute to 10 6 Pieces / ml; 1ml of spore suspension was inoculated into an Erlenmeyer flask containing 80ml of CM medium, and cultured at 28°C and 180rpm for 24h. The cultured hyphae are filtered with sterile three-layer lens cleaning paper, and the hyphae are washed 2-3 times with distilled water, and then collected, dried with absorbent paper, and the collected hyphae are extracted immediately with RNA extraction kit or first It was frozen in liquid nitrogen and then stored at -80°C for subsequent extraction. RNA extracted samples were immediately reverse transcribed to obtain single-stranded cDNA.

[0065] The reverse transcription product is used...

Embodiment 3

[0072] Example 3: Construction of TrPac1 gene knockout vector

[0073] A three-stage method was used to construct a gene knockout vector, that is, the upstream and downstream sequences of the TrPac1 gene with a length of 1000bp-2000bp were inserted into the two ends of the hygromycin resistance gene.

[0074] Firstly, the upstream and downstream fragments of TrPac1 gene were amplified separately from the Trichoderma reesei genome. Each fragment was about 1000 bp in length. The pGEM-T vector was connected to the upstream and the KpnI and SalI restriction enzymes were used for the upstream and downstream. HidIII and XbaI restriction endonuclease double restriction enzyme cut sites, after cutting from the T vector, first connect the upstream fragment to pBS-Hyr with the same restriction enzyme cut site, and then use HidIII and XbaI to cut the above vector. Connect the downstream fragment after digestion of the T vector to the restriction site to construct the gene knockout vector.

[...

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Abstract

The invention discloses a transcriptional regulation factor derived from trichoderma reesei and a coding gene and application of the transcriptional regulation factor, and relates to an environment pH responsive transcriptional regulation factor TrPac1 full gene cloned from the trichoderma reesei, and a complementary deoxyribonucleic acid (cDNA) sequence. Nucleotide sequences are shown as SEQ ID No.1 and SEQ ID No.2, and a coded amino acid sequence is shown as SEQ ID No.3. The invention also provides a recombinant expression vector and a recombinant host cell containing the pH transcriptional regulation factor TrPac1 cDNA sequence, and application of the pH transcriptional regulation factor TrPac1 cDNA sequence to regulation of growth and development or stress resistance of the trichoderma reesei.

Description

Technical field [0001] The present invention relates to a transcription control factor isolated from fungi, in particular to an environmental pH response transcription control factor TrPac1 cloned from Trichoderma reesei and its cDNA sequence. Recombinant expression vectors of regulatory factor cDNA sequences and recombinant host cells, and their applications in regulating the growth and development and stress resistance of Trichoderma reesei belong to the field of separation and application of fungal transcription regulatory factors. Background technique [0002] Trichoderma reesei (Trichoderma reesei) is a mesophilic saprophytic fungus that exists widely in nature. It was originally isolated from cotton canvas during World War II (Mandels et al., 1957). Trichoderma reesei is an industrially important cellulase-producing strain, and it is also a model strain used to study the regulation mechanism of cellulase and hemicellulase gene expression. Trichoderma reesei has a relativel...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N15/63C12N15/80C12N1/15C12R1/885
Inventor 赫荣琳马立娟张东远陈树林
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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