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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Authors: | Maier Frank J Malz Sascha Lösch Anke P Lacour Thierry Schäfer Wilhelm |
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Affiliation: | 1. College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China;2. Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China;3. College of Agricultural and Biotechnology, China Agricultural University, Beijing 100193, China;4. School of Resources and Environmental, Anhui Agricultural University, Hefei 230036, China;5. Institute of Resources Environmental and Detection Technology, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Huhhot 010031, China;6. College of Agronomy, Shenyang Agricultural University, Shenyang 110866, China;7. Wuchuan Scientific Observing and Experimental Station of Agro-Environment, Ministry of Agriculture Wuchuan 011700, China |
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Abstract: | 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 800bp and was linearized in the middle. Using a genomic sequence of approximately 500bp 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 400bp 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. |
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Keywords: | Fusarium graminearum Gibberella zeae Homologous recombination Gene disruption |
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