Regulatory element for heterologous protein production in the fruiting body of filamentous fungi

A technology of regulatory elements and filamentous fungi, applied in fungi, using vectors to introduce foreign genetic material, sugar derivatives, etc., can solve problems such as different promoter regions

Inactive Publication Date: 2013-06-26
INTREXON CORP (US)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it was found that although the gpd genes in basidiomycetes are highly similar, the promoter regions of these genes are significantly different

Method used

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  • Regulatory element for heterologous protein production in the fruiting body of filamentous fungi
  • Regulatory element for heterologous protein production in the fruiting body of filamentous fungi
  • Regulatory element for heterologous protein production in the fruiting body of filamentous fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Identification of the highly expressed lectin gene sequence from Agaricus bisporus

[0076] Using the 2-D protein profile of total soluble protein, it was determined that the lectin gene was highly expressed in the fruiting bodies of Agaricus bisporus. The selected fruiting bodies (commercial intermediate hybrid strains) are close to maturity, but have no exposed gills. The newly harvested fruit body tissues were frozen in liquid nitrogen, ground with a mortar and pestle, extracted with cold acetone, 20% trichloroacetate and 0.2% dithiothreitol, and submitted to Alphalyse Inc. (Palo Alto, CA) for 2-D protein profiling. The resulting gel image is shown in figure 1 in.

[0077] figure 1 .2-D Gel Analysis of Agaricus bisporus Fruit Body Protein

[0078] According to the manufacturer’s recommendation, use Invitrogen’s IPG drystrips for one-dimensional isoelectric focusing and the NuPAGE precast gel system for two-dimensional separation to perform protein separation o...

Embodiment 2

[0080] Example 2: Isolation and sequencing of the 5'upstream DNA region of the lectin gene containing the promoter

[0081] Using Genome Walking (APAgene TM Gold Genome Walking Kit, BIO S&T Inc. Montreal, QC, Canada) was used to isolate the 5'upstream DNA region of the genome of the lectin gene. According to the lectin cDNA sequence, the following primers were designed: lectin-F1 (5'-TTCGTTCAACGGGACGGAAGAAGCCTTT-3') and lectin-F2 (5'-TCTGGTAGACGCGAATGCTGATGGTGTA-3') (Crenshaw et al., 1995), and used for genome steps shift. The AquaGenomic Kit (MultiTarget Pharmaceuticals LLC, Salt Lake City, Utah) was used to extract the genomic DNA of Agaricus bisporus and used as the basis of APAgene TM The protocol provided by Gold Genome Walking Kit is a template for genome walking PCR. In short, add 4 aliquots of 15-μl PCR reaction mixture to PCR tubes labeled A, B, C, and D. Use 3x APAgene Gold Buffer I for tubes A and B, and for tube C And D use APAgene TM Gold buffer II. 15μl pr...

Embodiment 3

[0084] Example 3: Demonstrating the ability of the lectin promoter to direct the expression of the GUS reporter gene in Agaricus bisporus

[0085] Construction of the binary vector pAGN-750( figure 2 ) To test the ability of the lectin promoter (called pLctn) to drive the expression of the β-glucuronidase (GUS) reporter gene in Agaricus bisporus. Using primers Lctn-BglII (5'-AGCTTAGATCTGAACACGCG TCGTTTACCTCC-3') and Lctn-NcoI (5'-GTAAGTCCATGGTCTGCTCAACGTTATGAGTTAGTTG-3'), pLctn was amplified from pAGN-745. The restriction enzyme sites BglII and NcoI were incorporated into the primers to facilitate cloning. The PCR product was digested with BglII / NcoI, and the digested product was used to replace the BglII-NcoI fragment in the GUS expression vector pAGN-030 to produce pAGN-750. The sequence in pAGN-750 was confirmed by sequencing. Following the transformation method described by Chen et al. (2000), pAGN-750 was introduced into Agrobacterium tumefaciens and then Agaricus bisporu...

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Abstract

The present invention provides compositions and methods for regulating expression of nucleotide sequences in fungi. Compositions are novel nucleotide sequences for a tissue preferred promoter isolated from the Agaricus bisporus lectin gene. The sequences drive expression preferentially to fruit body tissue. A method for expressing a nucleotide sequence in fungi using the regulatory sequences disclosed herein is provided. The method comprises transforming a fungal cell to comprise a nucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed fungus from the transformed cell.

Description

Technical field [0001] The present invention relates to the field of molecular biology, in particular to the field of regulation of fungal gene expression. The new composition and method can be used to improve fungal species through recombinant techniques known to those skilled in the art, such as improving pathogen, pest and insecticide resistance, yield and quality, extending production shelf life, and improving cooking, nutritional and medical value Etc., and to improve the industrial production of heterologous proteins. Background technique [0002] About 40% of commercially available enzymes are derived from filamentous fungi. Lowe, Handbook of Applied Mycology. Fungal Biotechnology (eds.) Arora, D.K. Elander, R.P. & Mukerji, K.G. 681-708 (Marcel Dekker, New York; 1992). These enzymes are usually produced by species of Aspergillus and Trichoderma. Because they secrete large amounts of protein into the medium, they can grow during large-scale fermentation, and they are gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C07H21/04
CPCC07K14/37C12N15/80
Inventor Z·石J·Q·威尔金森D·S·瓦尔特斯C·P·罗曼
Owner INTREXON CORP (US)
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