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Recombinant expression vector for changing trichoderma reesei mycelium morphology and application thereof

A technology of Trichoderma reesei and expression vectors, applied in the field of agricultural biology, can solve the problems of poor oxygen-dissolving ability of Trichoderma reesei and unsatisfactory protein production, and achieve improved protein secretion ability, high interference efficiency, and high construction efficiency Effect

Active Publication Date: 2019-01-04
INST OF ANIMAL SCI CAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the problems that the existing Trichoderma reesei has poor oxygen dissolving capacity and protein production cannot meet the needs of industry, the present invention provides a recombinant expression vector for changing the mycelium morphology of Trichoderma reesei

Method used

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  • Recombinant expression vector for changing trichoderma reesei mycelium morphology and application thereof
  • Recombinant expression vector for changing trichoderma reesei mycelium morphology and application thereof
  • Recombinant expression vector for changing trichoderma reesei mycelium morphology and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1. Obtain recombinant Trichoderma reesei

[0032] 1. Construction of recombinant plasmids pAPA-pdc1P-barA1-eno1P, pAPA-pdc1P-barA2-eno1P

[0033] Digest the pAPA-plasmid with NotI and AscI, electrophoresis, recover the linearized plasmid vector, and then connect it with pdc1 promoter, barA1 gene and eno1 promoter by homologous recombination. Transform Escherichia coli Trans1-T1 competent cells, spread on LB medium, and culture at 37°C for 12 hours. Colony PCR was performed on the coliform colonies grown on the plate to identify whether the three fragments had been ligated to pAPA. The coliform colony identified as positive by PCR was picked and inoculated in 3 ml LB medium (containing 100 μg / ml ampicillin), cultured overnight at 37° C. with shaking at 180 rpm, and the plasmid was extracted. The correctly sequenced plasmid was named pAPA-pdc1P-barA1-eno11P.

[0034] 2. Transformation of Trichoderma reesei

[0035] Trichoderma reesei SUS1 was inoculated on ...

Embodiment 2

[0039] Example 2. SUS1, barA1-i and barA2-i recombinant transformant hyphal morphology observation

[0040] 1. Morphological observation of mycelium on solid plate

[0041] Inoculate 1.0 μL 2×10 7 Spores of Trichoderma reesei SUS1 and barA1, barA2 transformant strains to MM+2% glucose, MM+2% xylan, MM+2% lactose, MM+2% cellobiose, MM+2% xylose, In MM+2% glycerol, MM+2% microcrystalline cellulose and PDA medium, each sample was done in triplicate. Cultivate statically at 28°C, observe and record the growth status of mycelia and spores every 12 hours, and measure the length of mycelia.

[0042] Such as figure 2 As shown, in the solid medium of different carbon sources, the representative strains bar1-5 and bar2-15 of the recombinant bacteria barA1-i and barA2-i compared with the original strain SUS1, the sporulation and hyphal growth occurred Changes, the sporulation rate in PDA medium was significantly slowed down, and on glucose medium, its mycelial growth rate was simila...

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Abstract

The invention belongs to the technical field of agricultural ecology, and particularly relates to a recombinant expression vector for changing trichoderma reesei mycelium morphology and application thereof. The recombinant expression vector comprises gene barA1 and / or gene barA2, and is applied to converting trichoderma reesei to obtain recombinant trichoderma reesei with mycelum morphology changed, thereby improving protein expression of trichoderma reesei and cellulase activity.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a recombinant expression vector for changing the hyphae shape of Trichoderma reesei and its application. Background technique [0002] Filamentous fungi have the unique characteristic of mycelial polar growth, which can produce a kind of heat-stable antifungal factor compound, which can regulate the positioning of septum and formate at the end of mycelium, and then affect the polarity of mycelium. grow. Trichoderma reesei is the main industrial filamentous fungus producing cellulase today. Its cellulase has high yield, good stability and is easy to separate and purify. Trichoderma reesei not only has the characteristics of filamentous fungi with polar mycelial growth, but also has the characteristics of aerobic fungi, and its enzyme production process is closely related to the respiratory metabolism of mitochondria. During the liquid fermentation process, o...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/885
CPCC12N15/113C12N15/80C12N2800/10
Inventor 姚斌苏小运李梦珠罗会颖黄火清王亚茹柏映国涂涛王苑孟昆
Owner INST OF ANIMAL SCI CAAS
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