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Strong promoter from trichoderma reesei as well as expression vector and application thereof

A strong promoter, Trichoderma reesei technology, applied in the field of strong promoters, can solve the problem of less research on strains

Inactive Publication Date: 2014-06-25
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

[0004] At present, the research on Trichoderma reesei mainly focuses on the induced expression of genes to obtain the target products, especially in various secreted target proteins, and there are few studies on the species itself.

Method used

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  • Strong promoter from trichoderma reesei as well as expression vector and application thereof
  • Strong promoter from trichoderma reesei as well as expression vector and application thereof
  • Strong promoter from trichoderma reesei as well as expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Isolation of the promoter RPL from Trichoderma reesei

[0039] Genomic DNA was extracted from Trichoderma reesei strain QM9414 (preserved in the 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.

[0040] Extraction of Genomic DNA from Reproductive Hyphae of Trichoderma reesei

[0041] Wash the spores of Trichoderma reesei strain QM9414 grown on the PDA plate with sterilized water, filter them with sterilized three-layer lens-cleaning paper, and dilute the concentration to 10 6 Individuals / ml; take 1ml of spore suspension and inoculate in a conical flask containing 80ml of CM medium, culture at 28°C and 180rpm for 24h. Filter the cultured mycelium with sterilized three-layer lens-cleaning paper, wash the mycelium with distilled water for 2-3 times, collect it, dry it with absorbent paper, and freeze it at -20°C. Grind the...

Embodiment 2

[0051] Embodiment 2: corresponding to the acquisition of Trichoderma reesei RPL terminator

[0052] Primers were designed according to the information in the Trichoderma reesei genome database, and the RPL terminator of the target fragment was obtained by high-fidelity PCR. The primer sequences are as follows:

[0053] TRPLp1: 5'-ATtctagaCGAGGAGCTGCTTTCTTAT-3'

[0054] TRPLp2: 5'-AAgtcgacTTTCTCGTACTGGCAACA-3'

[0055] The lowercase font in the primer sequence indicates the corresponding restriction site, XbaI was introduced into the upstream primer, and Sali was introduced into the downstream primer.

[0056] The PCR reaction conditions, reaction system, and reaction process were the same as the reaction conditions, reaction system, and reaction process of the PCR amplification of the promoter RPL in Example 1.

[0057] The PCR product was recovered and purified by agarose gel electrophoresis, 3 μl of the RPL terminator gene product was recovered from the PCR gel, 1 μl of t...

Embodiment 3

[0058] Embodiment 3: Construction contains the expression vector of Trichoderma reesei RPL promoter and terminator

[0059] figure 1 It is the process of constructing the expression vector containing Trichoderma reesei RPL promoter and terminator. As can be seen from the figure, the expression vector construction process is as follows: after the original pCAMBIA1300 plasmid was cut with the restriction endonucleases KpnI and BamHI double restriction enzymes contained in the RPL promoter, combined with the RPL promoter (SEQ ID No. .1) connected, the connection system is as described in the connection condition system of the recombinant vector in the embodiment 1 or 2, and the expression vector containing the RPL promoter of Trichoderma reesei is constructed. The ligation product was transformed into Escherichia coli DH-5α, and the Escherichia coli DH-5α containing the recombinant plasmid vector was smeared on a plate containing kanamycin resistance for screening, the transfor...

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Abstract

The invention discloses a strong promoter from trichoderma reesei and application of the strong promoter. The strong promoter and a terminator corresponding to the strong promoter are obtained by being separated from trichoderma reesei and cloned, wherein the nucleotide sequences of the strong promoter and the terminator are as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The invention further provides a recombined expression vector including the strong promoter and the terminator corresponding to the strong promoter as well as a converter including the recombined expression vector. The invention further discloses the application of the strong promoter in the protein homogenous or heterogeneous expression as well as gene positioning.

Description

technical field [0001] The present invention relates to a strong promoter for fungal protein expression, in particular to a strong promoter isolated and cloned from Trichoderma reesei, a plasmid vector containing the strong promoter and a terminator, and a plasmid containing the plasmid vector Transformants, and their application in Trichoderma reesei protein homologous or heterologous expression, the present invention belongs to the field of isolation and application of promoters derived from Trichoderma reesei. Background technique [0002] Trichoderma reesei (Trichoderma reesei) is a mesophilic saprophytic fungus that exists widely in nature, and it can secrete a variety of enzymes, including cellulase, hemicellulase, protease and amylase, especially the high content of cellulase , the secreted cellulase has been widely used in feed, sugar, chemical fiber, paper, and other industries for many years (Feed Industry, 2003, 24 (1)). In recent years, with the emergence of ene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/15C12N15/80C12R1/885
Inventor 赫荣琳杨宗鑫马立娟张东远陈树林
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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