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Recombinant expression vector for rapidly increasing cellulase activity of Trichoderma reesei and application thereof

A technology of Trichoderma reesei and expression vector, which is applied in the field of agricultural biology and can solve the problems of not being able to significantly improve the effect of transcriptional activation

Inactive Publication Date: 2019-01-18
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, the overexpression of the above-mentioned transcriptional activators does not significantly improve the transcriptional activation effect

Method used

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  • Recombinant expression vector for rapidly increasing cellulase activity of Trichoderma reesei and application thereof
  • Recombinant expression vector for rapidly increasing cellulase activity of Trichoderma reesei and application thereof
  • Recombinant expression vector for rapidly increasing cellulase activity of Trichoderma reesei and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 obtains recombinant Trichoderma reesei

[0033] 1. Construction of recombinant plasmids pPpdc1-OExyr1 and pPcbh1-OExyr1

[0034]The promoters of cbh1 and pdc1 were ligated with xyr1-Txyr1 and AmpR-Pyr4-AmpR respectively according to the instructions of the seamless splicing kit. The ligated fragments were transformed into Trans1 competent cells, and positive transformants were obtained by screening on a solid LB medium plate containing 100 μg / ml ampicillin. The clones were picked and cultured overnight in 3 ml liquid LB medium containing 100 μg / ml ampicillin, the plasmids were extracted, sequenced and verified to obtain recombinant plasmids pPpdc1-OExyr1 and pPcbh1-OExyr1, ready for transformation.

[0035] 2. Transformation of pPpdc1-OExyr1 and pPcbh1-OExyr1 into Trichoderma reesei

[0036] Trichoderma reesei TU-6 was inoculated on a potato medium (PDA) plate, and cultured statically at 28°C for 7 days until it produced spores, and the spores were scrape...

Embodiment 2

[0045] Example 2 Identification of Enzyme Activity and Transcription Abundance of Recombinant Trichoderma reesei

[0046] 1. Determination of cellulase activity

[0047] For the starting strain TU-6 and each transformant, after pre-determining the cellulase activity of the transformants, respectively select a transformant of the transformed plasmid Ppdc1-OExyr1 and plasmid Pcbh1-OExyr1 with high cellulase activity to measure the following data : Exo-cellulase activity (CBHI activity), endo-cellulase activity (EG activity), overall cellulase activity (filter paper activity).

[0048] (1) Determination of exocellulase activity:

[0049] Using 4-methylumbelliferyl β-D-lactoside as a substrate, weigh 15 mg MUL, dissolve it in 500 μL DMSO, and transfer it to 30 ml citric acid buffer (pH 5.0, 50 mM). Mix 25 μL of appropriately diluted enzyme solution, 200 μL of MUL and 25 μL of glucose (1M), this is the experimental group without adding cellobiose. In the mixed solution, glucose ...

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Abstract

The invention belongs to the field of agricultural biotechnology, in particular to a recombinant expression vector for rapidly increasing cellulase activity of Trichoderma reesei and application thereof. The invention uses positive feedback control mechanism to place xyr1 gene under promoter cbh1, pdc1, constructs expression plasmid vector and transforms Trichoderma reesei. Overexpression of xyr1activated both cellulase and self-expression, forming a positive feedback regulatory network, and the cellulase and hemicellulase activities of the positive transformants were significantly increased.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a recombinant expression vector for rapidly improving the enzyme activity of cellulase produced by Trichoderma reesei and its application. Background technique [0002] The plant cell wall is mainly composed of cellulose, hemicellulose and lignin, which degrade the polysaccharides in the plant cell wall, especially the main component cellulose, into fermentable oligosaccharides and monosaccharides For example, glucose requires a complex cellulase enzyme system to act synergistically. [0003] As one of the main filamentous fungi, Trichoderma reesei has a strong ability to secrete cellulase. The cellulase system of Trichoderma reesei includes two exo-cellulase, five endo-cellulase, one β-glucosidase and three lytic polysaccharide monooxygenases (LPMOs, namely former GH61 family member). The regulation of the secretion and expression of these cellulases is v...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12N9/42C12R1/885
CPCC12N9/2437C12N9/2445C12N15/80C12N2830/002C12Y302/01004C12Y302/01021C12Y302/01091
Inventor 姚斌苏小运孙先花罗会颖黄火清王亚茹柏映国涂涛王苑孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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