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Recombinant vector and recombinant bacterium of Trichoderma reesei beta-glucosaccharase gene BGL1, and expression of Trichoderma reesei beta-glucosaccharase gene BGL1 in recombinant bacterium

A glucosidase and Trichoderma reesei technology, applied in the field of bioengineering, can solve the problems of low β-glucosidase content, loss of β-glucosidase activity, unreachable, etc., so as to improve degradation efficiency and solve energy problems. Crisis, the effect of increasing production

Inactive Publication Date: 2012-10-10
TIANJIN UNIV
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AI Technical Summary

Problems solved by technology

[0004] In the process of cellulase hydrolyzing cellulose, there are mainly the following problems at present: (1) in the strains producing cellulase, the content of β-glucosidase is very low, far below the level of practical application; (2) During the reaction, due to thermal inhibition, most of the activity of β-glucosidase will be lost
[0006] Cellulose hydrolysis is limited by the low production of β-glucosidase in native Trichoderma reesei strains

Method used

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  • Recombinant vector and recombinant bacterium of Trichoderma reesei beta-glucosaccharase gene BGL1, and expression of Trichoderma reesei beta-glucosaccharase gene BGL1 in recombinant bacterium
  • Recombinant vector and recombinant bacterium of Trichoderma reesei beta-glucosaccharase gene BGL1, and expression of Trichoderma reesei beta-glucosaccharase gene BGL1 in recombinant bacterium
  • Recombinant vector and recombinant bacterium of Trichoderma reesei beta-glucosaccharase gene BGL1, and expression of Trichoderma reesei beta-glucosaccharase gene BGL1 in recombinant bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning of Example 1 Trichoderma reesei (Trichoderma reesei) β-glucosidase gene

[0036] The total RNA of Trichoderma reesei CICC 13052 was extracted with Trizol reagent according to the instructions of the kit.

[0037] Get 2ug of total RNA, reverse-transcribe it into cDNA (Promega Company) with a reverse transcription kit, use this cDNA as a template, and use the nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing Carry out PCR for upstream primer and downstream primer, amplify the Trichoderma reesei β-glucosidase gene BGL1 gene of 2235bp shown in SEQ ID NO.3 in the sequence listing (see figure 1 ). Reclaim the PCR product, be connected to TA carrier pGEM-T (Promega company) and obtain new plasmid, confirm through sequencing that this plasmid contains Trichoderma reesei β-glucosidase gene BGL1, and the plasmid is named as pGEM-BGL1, promptly obtains containing Richteri Plasmid pGEM-BGL1 of the BGL1 gene of Trichoderma. build strategy se...

Embodiment 2

[0038] Example 2 Construction of Trichoderma reesei β-glucosidase gene BGL1 to Pichia pastoris expression vector

[0039] Using the plasmid pGEM-BGL1 as a template, using the nucleotide sequence shown in SEQ ID NO.4 in the sequence listing as an upstream primer, and using the nucleotide sequence shown in SEQ ID NO.5 in the sequence listing as a downstream primer for PCR amplification, Obtain the 2198bp fragment shown in SEQ ID NO.6 in the sequence table, connect the 2198bp fragment with the TA vector pGEM-T with T4 ligase, transform the transformant obtained by Escherichia coli TOP10F', and confirm the plasmid in the transformant by sequencing It is formed by connecting Trichoderma reesei β-glucosidase gene BGL1 and pGEM-T, and the new plasmid obtained by transformation is named pTBGL1, and the plasmid pTBGL1 is extracted from the above-mentioned transformants, and then obtained from Pichia pastoris The pPICZalpha C plasmid was extracted from the Escherichia coli expressing th...

Embodiment 3

[0040] Example 3 Trichoderma reesei β-glucosidase gene BGL1 integrated into Pichia pastoris chromosome:

[0041] Take 10 μg (you can also choose 15 μg) of the pPICZalpha C-BGL1 plasmid, use PmeI enzyme to cut into the 5713bp fragment shown in the linear SEQ ID NO.9, after phenol / chloroform extraction, ethanol precipitation linear SEQ ID NO.9 After drying, dissolve the DNA fragment shown in sterile water to make a DNA solution with a DNA concentration of 1 μg / μL (or 0.5 μg / μL);

[0042] Prepare the competent cells of Pichia pastoris host strain X-33 by referring to the electroporation transformation preparation yeast competent cell method in the refined Molecular Biology Experiment Guide (Fourth Edition) (P512-513), and 80 μL Bath Mix the competent cells of Pichia host strain X33 with 10 μL of 1 μg / μL (or 0.5 μg / μL) linear DNA solution, and then transfer to 4°C (or any value between 0-4°C) ) into the pre-cooled electrode cup, ice bath for 5min, voltage 1500V, electric shock wi...

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Abstract

The invention discloses a recombinant vector and recombinant bacterium of Trichoderma reesei beta-glucosaccharase gene BGL1, and expression of the Trichoderma reesei beta-glucosaccharase gene BGL1 in the recombinant bacterium. The recombinant vector containing the Trichoderma reesei beta-glucosaccharase gene BGL1 is constructed by the following steps: (1) carrying out PCR (Polymerase Chain Reaction) amplification on the Trichoderma reesei beta-glucosaccharase gene BGL1, and connecting to a TA vector; and (2) constructing the Trichoderma reesei beta-glucosaccharase gene to a pichia pastoris expression vector. The recombinant bacterium disclosed by the invention can effectively express the target protein Trichoderma reesei beta-glucosaccharase BGL1. The invention overcomes the defect that agreat deal of beta-glucosaccharase can not be purified from Trichoderma reesei in the prior art. Lots of waste lignocellulose exists in the natural world. The method provided by the invention can increase the yield of beta-glucosaccharase, and has very important meanings in enhancing degradation efficiency of cellulose, efficiently producing energy from waste cellulose and solving the problem of energy crisis at present.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a recombinant vector containing Trichoderma reesei β-glucosidase BGL1 gene, a recombinant Pichia pastoris containing Trichoderma reesei β-glucosidase BGL1 gene and Induced expression of Trichoderma reesei β-glucosidase BGL1 gene in recombinant Pichia pastoris. Background technique [0002] Cellulose, the main component of plant cell walls, is the largest renewable carbon source on Earth. The use of biotransformation to produce fuel ethanol and other chemicals is of great significance for the current human beings to solve problems such as energy crisis, food shortage, and environmental pollution. [0003] Cellulase degrading cellulose is a multi-enzyme system comprising endoglucanase, exocellobiohydrolase and β-glucosidase. Endoglucanase, exoglucanase and β-glucosidase, these three enzymes work together to degrade cellulose into glucose: endoglucanase cuts the long chains of cellul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N1/19C12N9/42C12R1/885C12R1/84
Inventor 马媛媛邹少兰洪解放张鲲井欣张敏华
Owner TIANJIN UNIV
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