A METHOD FOR EASY ISOLATION OF PROMOTER FRAGMENTS FROM PROMOTER-PROBE LIBRARIES OF FILAMENTOUS FUNGI

被引：5

作者：

WELTRING, KM ^{[1
]}

机构：

[1] UNIV MUNSTER,BOT GARDENS,D-48149 MUNSTER,GERMANY

来源：

CURRENT GENETICS | 1995年 / 28卷 / 02期

关键词：

PROMOTER; FUNGI; FUSARIUM; TRANSFORMATION;

D O I：

10.1007/BF00315787

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

Two genomic fragments capable of driving the expression of the hygromycin B resistance gene (hph) were isolated from the phytopathogenic ascomycete Gibberella pulicaris (anamorph Fusar ium sambucinum) using a ''promoter-probe library'' strategy. Two libraries consisting of random, 0.5-2.0-kb fragments of genomic DNA inserted 5' of a promoterless hph gene were constructed and used for transformation of G. pulicaris. Both libraries transformed G, pulicaris at a low frequency. Transformants tolerated up to 800 mu g/ml of hygromycin B, while untransformed colonies were inhibited completely by 50 mu g/ml of the antibiotic. Plasmids were re-isolated from transformants by simply digesting, the genomic DNA with KpnI, which cuts once in the polylinker 5' to the insert, and transforming E. coli with the re-ligated DNA. The recovered plasmids transformed G. pulicaris with a frequency of up to 4.4 transformants/mu g of DNA. Both promoter fragments were sequenced and found to contain TATA and CAAT boxes as well as CT-rich sequences. This method makes it possible to easily isolate many fragments with promoter activity from filamentous fungi, and should facilitate the investigation of the promoter structures necessary for the expression of fungal genes.

引用

页码：190 / 196

页数：7

共 25 条

[1]

Ballance D.J., Transformation system for filamentous fungi and an overview of fungal gene structure, Molecular and industrial mycology, pp. 1-29, (1990)

[2]

Cullen D., Leong S.A., Wilson L.J., Henner D.J., Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph, Gene, 57, pp. 21-26, (1987)

[3]

Desjardins A.E., Beremand M.N., A genetic system for trichothecene toxin production in Gibberella pulicaris (Fusarium sambucinum), Phytopathology, 77, pp. 678-683, (1987)

[4]

Desjardins A.E., Gardner H.W., Genetic analysis in Gibberella pulicaris: rishitin tolerance, rishitin metabolism, and virulence on potato tubers, Molecular Plant-Microbe Interactions, 2, pp. 26-34, (1989)

[5]

Desjardins A.E., Gardner H.W., Virulence of Gibberella pulicaris on potato tubers and its relationship to a gene for rishitin metabolism, Phytopathology, 81, pp. 429-435, (1991)

[6]

Desjardins A.E., Gardner H.W., Weltring K-M, Detoxification of sesquiterpene phytoalexins by Gibberella pulicaris (Fusarium sambucinum) and its importance for virulence on potato tubers, J Ind Microbiol, 9, pp. 201-211, (1992)

[7]

Gatignol A., Dassain M., Tiraby G., Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance, Gene, 91, pp. 35-41, (1990)

[8]

Goodey A.R., Doel S.M., Piggott J.R., Watson M.E.E., Zealey G.R., Cafferkey R., Carter B.L.A., The selection of promoters for the expression of heterologous genes in the yeast Saccharomyces cerevisiae, Mol Gen Genet, 204, pp. 505-511, (1986)

[9]

Horwitz Z., Loeb L.A., Promoters selected from random sequences, Proc Natl Acad Sci USA, 83, pp. 7405-7409, (1986)

[10]

Jungehulsing U., Arntz C., Smit R., Tudzynski P., The Claviceps purpurea glyceraldehyd-3-phosphate dehydrogenase gene: cloning, characterization, and use for the improvement of a dominant selection system, Current Genetics, 25, pp. 101-106, (1994)

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