The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation

1989 ◽  
Vol 218 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Shiela E. Unkles ◽  
Edward I. Campbell ◽  
Yolanda M. J. T. de Ruiter-Jacobs ◽  
Martien Broekhuijsen ◽  
Janet A. Macro ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Nitrate Assimilation ◽  
Fungal Transformation ◽  
Selection System ◽  
Transformation System ◽  
Homologous Transformation

scholarly journals Development of an Efficient and Stable Transformation System for Aspergillus Oryzae Based on the pyrG Gene

2020 ◽  
Author(s):  
Caixia Zhou ◽  
Yujun Wan ◽  
Huipeng Yao ◽  
Hui Chen ◽  
Yirong Xiao ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Transformation Method ◽  
Stable Transformation ◽  
Fungal Transformation ◽  
Transformation System ◽  
Efficient Expression ◽  
The Stability ◽  
Green Fluorescent

Abstract Background Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. An efficient and stable transformation system is the key to the successful expression of the gene of interest in A. oryzae.Results To improve the expression efficiency of the gene of interest in A. oryzae, we constructed the uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG by Ultraviolet (UV) mutagenesis of pyrG gene deletion which would be used as a host for further transformation. In addition, a novel and efficient expression vector pBC-hygro.4 was constructed, including the pyrG cassette gene, His-Tag, amyB promoter and terminator,and green fluorescent protein GFP marker. pBC-hygro.4 transformed A. oryzae RIB40ΔpyrG efficiently via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was tested by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, we successfully obtained a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG. At the same time, the developed vectors are fully functional for heterologous expression of the GFP fluorescent proteins in the A. oryzae RIB40ΔpyrG.Conclusion Our work provides a new method that can be applied to other filamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.

Development of a Homologous Transformation System forAspergillus aculeatusBased on thesCGene Encoding ATP-Sulfurylase

2009 ◽  
Vol 73 (5) ◽  
pp. 1197-1199 ◽  
Author(s):  
Hiromi ADACHI ◽  
Shuji TANI ◽  
Shin KANAMASA ◽  
Jun-ichi SUMITANI ◽  
Takashi KAWAGUCHI
Keyword(s):  
Transformation System ◽  
Atp Sulfurylase ◽  
Homologous Transformation

Development of a fungal transformation system based on selection of sequences with promoter activity

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

scholarly journals Development of a fungal transformation system based on selection of sequences with promoter activity.

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305 ◽  
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

1987 ◽  
Vol 206 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Wim van Hartingsveldt ◽  
Ineke E. Mattern ◽  
Cora M. J. van Zeijl ◽  
Peter H. Pouwels ◽  
Cees A. M. J. J. van den Hondel
Keyword(s):  
Aspergillus Niger ◽  
Transformation System ◽  
Homologous Transformation

scholarly journals The Naturally Evolved EPSPS From Goosegrass Confers High Glyphosate Resistance to Rice

2021 ◽  
Vol 12 ◽  
Author(s):  
Chao Ouyang ◽  
Wei Liu ◽  
Silan Chen ◽  
Huimin Zhao ◽  
Xinyan Chen ◽  
...  
Keyword(s):  
Selection Pressure ◽  
High Efficiency ◽  
Rice Genome ◽  
Selection System ◽  
Transformation System ◽  
Wild Type ◽  
Promising Alternative ◽  
Two Generations ◽  
Massive Scale ◽  
Similar Growth

Glyphosate-resistant crops developed by the CP4-EPSPS gene from Agrobacterium have been planted on a massive scale globally, which benefits from the high efficiency and broad spectrum of glyphosate in weed control. Some glyphosate-resistant (GR) genes from microbes have been reported, which might raise biosafety concerns. Most of them were obtained through a hygromycin-HPT transformation system. Here we reported the plant source with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from goosegrass endowed rice with high resistance to glyphosate. The integrations and inheritability of the transgenes in the rice genome were investigated within two generations. The EiEPSPS transgenic plants displayed similar growth and development to wild type under no glyphosate selection pressure but better reproductive performance under lower glyphosate selection pressure. Furthermore, we reconstructed a binary vector pCEiEPSPS and established the whole stage glyphosate selection using the vector. The Glyphosate-pCEiEPSPS selection system showed a significantly higher transformation efficiency compared with the hygromycin-HPT transformation system. Our results provided a promising alternative gene resource to the development of GR plants and also extended the plant transformation toolbox.

Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae

2009 ◽  
Vol 46 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Y SHIMA ◽  
Y ITO ◽  
S KANEKO ◽  
H HATABAYASHI ◽  
Y WATANABE ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Transformation System
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