کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9278986 | 1593165 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
میکروبیولوژی و بیوتکنولوژی کاربردی
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چکیده انگلیسی
We cloned a polyketide synthase gene (pks12) from Fusarium graminearum, a devastating fungal pathogen of cereals. Transformation-mediated gene disruption led to an easily detectable albino phenotype of the disruptants. We used the disruption of the pks12 gene as a visible marker for transformation-mediated homologous recombination and optimized the transformation procedure to achieve a high rate of homologous recombination. In combination with the published genomic sequence data and the generation of expressed sequence tags (ESTs) for F. graminearum, this is a useful tool to investigate this important plant pathogen on a molecular level. Optimized transformation of F. graminearum resulted in at least 93% homologous recombination events when the homologous genomic DNA fragment in the vector had a size of approximately 800Â bp and was linearized in the middle. Using a genomic sequence of approximately 500Â bp in the transformation vector, 70% of the transformants still exhibited homologous recombination. On the contrary, no more than 10% homologous recombination events were observed when less than 400Â bp DNA fragments were used. We co-transformed F. graminearum with two different vectors. One vector harboured a DNA insert homologous to the pks12 gene, while the other vector consisted of the same vector backbone carrying the selection marker specific for F. graminearum. About 70% of the transformants had a disrupted pks12 gene, and all of these showed an integration of the second vector into the pks disruption vector. Therefore, the time-consuming construction of a single transformation vector can be avoided; furthermore, it is now easily feasible to express a gene construct at a defined and mutated genomic site.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: FEMS Yeast Research - Volume 5, Issues 6â7, April 2005, Pages 653-662
Journal: FEMS Yeast Research - Volume 5, Issues 6â7, April 2005, Pages 653-662
نویسندگان
Frank J. Maier, Sascha Malz, Anke P. Lösch, Thierry Lacour, Wilhelm Schäfer,