کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2181099 1095267 2010 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A Simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
A Simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells
چکیده انگلیسی

Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20 years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1 × 104 Hygromycin B resistant colony forming units (CFU) have been achieved on each 90 mm agar plate using 106 sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5 kb from the coding sequences at the 5′ UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Fungal Genetics and Biology - Volume 47, Issue 4, April 2010, Pages 279–287
نویسندگان
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