Constitutive expression cassette of Trichoderma reesei, expression vector, and recombinant strain and application thereof

A constitutive expression technology of Trichoderma reesei, applied in the field of genetic engineering research, can solve the problems of low expression level, unreported recombinant strains of Trichoderma reesei, unsuitable for industrial application process, etc., and achieve high protein expression level Effect

Inactive Publication Date: 2013-03-20
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when XYN Ⅱ is expressed under the control of T. reesei's own inducible promoter, the expression level is low, and it is mixed with cellulase, which is not suitable for some industrial applications
[0012] So far, the construction of a constitutive expression system using the promoter of the pdc gene of Trichoderma reesei has not been reported at home and abroad. The Trichoderma reesei constructed by this method can efficiently express xylanase without cellulase Recombinant strains have not been reported at home and abroad

Method used

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  • Constitutive expression cassette of Trichoderma reesei, expression vector, and recombinant strain and application thereof
  • Constitutive expression cassette of Trichoderma reesei, expression vector, and recombinant strain and application thereof
  • Constitutive expression cassette of Trichoderma reesei, expression vector, and recombinant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of Trichoderma reesei constitutive expression cassette and expression vector

[0041] 1. Extraction and inspection of Trichoderma reesei genomic DNA

[0042] Trichoderma reesei QM9414 (commonly used strain in this field) was inoculated on PDA medium, and cultured at 28°C for 7 days until the spores matured. Prepare an appropriate amount of spore suspension and inoculate it in a liquid seed medium, and culture it at 30° C. and 180 rpm for 2 days until the concentration of mycelium reaches 4-5 g / L for extraction of genomic DNA.

[0043] The formula of the above-mentioned liquid seed medium is as follows: after mixing 20.0g of glucose, 2.0g of peptone, 100ml of Mandels nutrient solution concentrate, 50ml of citric acid buffer solution (pH4.5, 1mol / L) and 1ml of Mandels trace element concentrate, use Dissolve in distilled water and make up to 1 L.

[0044] The formula of the above-mentioned Mandels nutrient solution concentrate is: after mixing 14g ...

Embodiment 2

[0062] Example 2 Expression of xylanase gene xyn2 by Trichoderma reesei constitutive expression system

[0063] 1. Isolation of xyn2 gene

[0064] The genomic DNA of Trichoderma reesei was used as a template, and the primers X1 and X2 in Table 1 were used for PCR amplification to obtain the xyn2 gene.

[0065] Upstream primer X1, the nucleotide sequence of which is shown in SEQ ID NO: 7, which contains the enzyme cutting site of Xba I;

[0066] The nucleotide sequence of the downstream primer X2 is shown in SEQ ID NO: 8, which contains a BamHI restriction site.

[0067] The amplified product was detected by agarose electrophoresis, and its molecular size was 805bp, which was in line with the expected result; by DNA sequence analysis, its sequence had 100% homology with the sequence published on GeneBank.

[0068] 2. Construction of Ppdc-XYN-Tpdc expression cassette

[0069] The xyn2 gene obtained above was digested with Xba Ⅰ and BamH Ⅰ and then connected to the expression ...

Embodiment 3

[0077] Example 3 Analysis of expression products of recombinant strain T.reesei-XYNII

[0078]The recombinant strain T.reesei-XYNII that grew well on the PDA plate containing hygromycin resistance was inoculated in Trichoderma reesei liquid seed medium, and after 48 hours of cultivation, it was transferred to recombinant T. reesei at an inoculation amount of 5%. In the mold expression medium, the expression of XYNII was analyzed after culturing for 144 hours.

[0079] The formula of above-mentioned Trichoderma reesei liquid seed is: 100ml / L Mandels nutrient solution concentrate, 1.0ml / L Mandels trace element concentrate, 10g / L glucose, 1.0g / L peptone, the citric acid buffer solution of 50ml / L ( pH4.5, 1mol / L), 1.0~2.0g / L Tween 80.

[0080] The formula of the above-mentioned recombinant Trichoderma reesei expression medium is: 100ml / L Mandels nutrient solution concentrate, 1.0ml / L Mandels trace element concentrate, 60g / L glucose, 5.0g / L peptone, 50ml / L citric acid buffer (pH4...

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Abstract

The invention which relates to the field of genetic engineering provides a constitutive expression cassette of Trichoderma reesei. The expression cassette comprises a foreign target gene, a promoter of a pyruvate decarboxylase gene (Ppdc) of Trichoderma reesei, and a terminator of the pyruvate decarboxylase gene (Tpdc) of Trichoderma reesei, wherein the nucleotide sequence of the Ppdc is represented by SEQ ID NO.1, and the nucleotide sequence of the Tpdc is represented by SEQ ID NO.2. The expression cassette which comprises the sequence of the Ppdc of Trichoderma reesei and the sequence of the Tpdc of Trichoderma reesei can express genes from animals, plant or fungi without induction, and can properly modify expression products, protein expression can be carried out with the highly efficient synthesis and secretion capability of Trichoderma reesei, the protein expression level is high, and excessive foreign proteins in the expression products can be avoided. A recombinant strain of the present invention can highly express xylanase without cellulose, and the xylanase has an important application in the papermaking industry.

Description

technical field [0001] The invention belongs to the field of genetic engineering research, in particular to a constitutive expression cassette of Trichoderma reesei and its application. Background technique [0002] Some filamentous fungi have a strong ability to synthesize and secrete proteins, and can glycosylate the expressed proteins, so they are a type of potential host bacteria for exogenous protein expression, commonly used Aspergillus nidulans , Aspergillus niger, Trichoderma reesei, Aspergillus awamori, etc. [0003] Trichoderma reesei is an efficient cellulase producer, and its cellulase system includes five endo-β-glucosidases (EG I, EG II, EG III, EG IV and EG V), two fiber Disaccharide hydrolases (CBH I and CBH II) and two cellobiases (BG I and BG II). Among them, the content of CBH Ⅰ is the highest, and its expression can reach 50% of the total amount of extracellular secreted proteins of Trichoderma reesei, and the total amount of synthesized and secreted pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63C12N1/15C12N9/42A61K38/47A23K1/16C12R1/885
Inventor 邢苗刘刚李俊鑫
Owner SHENZHEN UNIV
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