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Trichoderma reesei expression cassette, recombinant strain and application thereof

A technology of Trichoderma reesei and recombinant strains, which is applied in the field of genetic engineering, can solve the problems of low expression level, no report of Trichoderma reesei recombinant strains, and inability to apply to industrial application processes, and achieves the effect of high protein expression.

Inactive Publication Date: 2009-10-21
邢苗 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when XYNII 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
[0011] So far, the use of the promoter of the gpd gene of Trichoderma reesei to construct a constitutive expression system 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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  • Trichoderma reesei expression cassette, recombinant strain and application thereof
  • Trichoderma reesei expression cassette, recombinant strain and application thereof
  • Trichoderma reesei expression cassette, recombinant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The construction of embodiment 1 Trichoderma reesei constitutive expression vector

[0044] 1. Extraction and inspection of Trichoderma reesei genomic DNA

[0045] Trichoderma reesei QM9414 (commonly used strain in this field) was inoculated on PDA medium, and cultured at 28° C. for 7 days at a constant temperature 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.

[0046] The formula of above-mentioned liquid seed culture medium is: glucose 20.0g, peptone 2.0g, (NH 4 ) 2 SO 4 1.4g, KH 2 PO 4 2.0g, urea 0.3g, CaCl 2 0.3g, 100ml of Mandels nutrient solution concentrate, 50ml of citric acid buffer solution (pH4.5, 1mol / L) and 1ml of Mandels trace element concentrate were mixed, dissolved in distilled water and adjusted to 1L.

[0047] The formula of the above-mentioned ...

Embodiment 2

[0063] Example 2 Utilize Trichoderma reesei constitutive expression system to express green fluorescent protein

[0064] 1. Acquisition of the green fluorescent protein gene GFP

[0065] Using the plasmid pCAMBIA1302 (commercially available) as a template, the 750 bp green fluorescent protein GFP gene was amplified by PCR using primers F1 and F2.

[0066]The upstream primer F1, the nucleotide sequence of which is shown in SEQ ID NO: 7, which contains the enzyme cutting site of Xba I;

[0067] The nucleotide sequence of the downstream primer F2 is shown in SEQ ID NO: 8, which contains a Kpn I enzyme cutting site.

[0068] The length of the amplified product was 750bp, and the results of length and sequence analysis were in line with expectations.

[0069] 2.P gpd -GFP-T cbh1 Construction of expression cassettes

[0070] The gfp gene was connected to the expression vector pUC-PT through restriction enzyme cutting sites (Xba I and Kpn I) and ligase to obtain gpd -GFP-T cbh...

Embodiment 3

[0082] Example 3 Utilize Trichoderma reesei constitutive expression system to express xylanase gene xyn2

[0083] 1. Isolation of xyn2 gene

[0084] Genomic DNA of Trichoderma reesei was used as a template and primers X1 and X2 were used for PCR amplification to obtain xyn2 gene.

[0085] Upstream primer X1, the nucleotide sequence of which is shown in SEQ ID NO: 9, which contains the enzyme cleavage site of Xba I;

[0086] The downstream primer X2, whose nucleotide sequence is shown in SEQ ID NO: 10, contains a Kpn I enzyme cutting site.

[0087] The amplified product was detected by agarose electrophoresis, and its molecular size was 800bp, which was in line with the expected result; by DNA sequence analysis, its sequence had 100% homology with the sequence published on GeneBank.

[0088] 2.P gpd -XYN-T cbh1 Construction of expression cassettes

[0089] The xyn2 gene obtained above was digested with Xba I and Kpn I and connected with the expression vector pUC-PT that ha...

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Abstract

The invention discloses a Trichoderma reesei expression cassette, a recombinant strain and application thereof. The expression cassette includes Trichoderma reesei glyceraldehyde-3-phosphate dehydrogenase promoter, foreign target genes and Trichoderma reesei CBHI terminator. The expression cassette of the invention adopts the technical scheme that the sequence of the Trichoderma reesei glyceraldehyde-3-phosphate dehydrogenase gene promoter and the sequence of the Trichoderma reesei CBHI terminator form a constitutive promoter, genes of animals, vegetables or epiphyte can be expressed without induction, expression products can be properly modified, the efficient synthesis and exudation capability of the Trichoderma reesei can be utilized for high protein expression, and overmuch foreign proteins can be prevented from being generated in the expression products. The recombinant strain of the invention can efficiently express cellulose-free xylanase which has important using value in the paper manufacturing industry.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Trichoderma reesei expression cassette and its recombinant strain and application. Background technique [0002] Trichoderma reesei is an efficient cellulase producer, and its cellulase system includes five endo-β-glucosidases (EG I, EG II, EGIII, EGIV and EGV), two fiber Disaccharide hydrolases (CBH I and CBHII) and two cellobiases (BG I and BGII), among which CBH I has the highest content, and its expression can reach 50% of the total amount of extracellular secretory proteins of Trichoderma reesei, And under the condition of induction, the total amount of protein synthesized and secreted by Trichoderma reesei can reach 40g / L. [0003] The CBH I of Trichoderma reesei is encoded by the cbh1 gene of a single copy, thus it can be seen that the cbh1 promoter that regulates its expression is a strong filamentous fungal promoter, therefore, the Trichoderma reesei const...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N1/15C12N9/42C12P21/02A23K1/16C12R1/885A23K10/16
Inventor 邢苗刘刚康康李云
Owner 邢苗
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