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首页> 外文期刊>Plant Breeding and Seed Science >Cloning and heterologous expression of candidate DON-inactivating UDP-glucosyltranferases from rice and wheat in yeast
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Cloning and heterologous expression of candidate DON-inactivating UDP-glucosyltranferases from rice and wheat in yeast

机译：酵母中水稻和小麦的DON失活候选UDP-葡萄糖基转移酶的克隆和异源表达

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Fusarium graminearum and related species causing Fusarium head blight of cereals and ear rot of maize produce the trichothecene toxin and virulence factor deoxynivalenol (DON). Plants can detoxify DON to a variable extent into deoxynivalenol-3-O-glucoside (D3G). We have previously reported the DON inactivating glucosyltransferase (UGT) AtUGT73C5 from Arabidopsis thaliana (Poppenberger et al, 2003). Our goal was to identify UGT genes from monocotyledonous crop plants with this enzymatic activity. The two selected rice candidate genes with the highest sequence similarity with AtUGT73C5 were expressed in a toxin sensitive yeast strain but failed to protect against DON. A full length cDNA clone corresponding to a transcript derived fragment (TDF108) from wheat, which was reported to be specifically expressed in wheat genotypes containing the quantitative trait locus Qfhs.ndsu-3BS for Fusarium spreading resistance (Steiner et al, 2009) was reconstructed. Only cDNAs with a few sequence deviations from TF108 could be cloned. However, toxin sensitive yeast strains expressing this wheat UGT cDNA did not show a resistant phenotype. The main difficulty in generating full length cDNAs for functional validation by heterologous expression in yeast is the enormous number of the UGT superfamily members in plants, with 107 UGT genes plus some pseudogenes in Arabidopsis thaliana and about 150 putative UGT genes in grasses. We conclude that neither sequence similarity nor inducibility are good predictors of substrate specificity.
机译：禾谷镰刀菌和引起谷类镰刀菌枯萎病的相关物种和玉米的耳腐产生了天花粉毒素和毒力因子脱氧雪腐烯酚（DON）。植物可以将DON在不同程度上解毒为脱氧雪茄烯-3-O-葡萄糖苷（D3G）。我们先前已经报道了来自拟南芥的DON失活葡萄糖基转移酶（UGT）AtUGT73C5（Poppenberger等，2003）。我们的目标是从具有这种酶活性的单子叶农作物中鉴定UGT基因。在毒素敏感的酵母菌株中表达了两个与AtUGT73C5具有最高序列相似性的水稻候选基因，但未能防御DON。重建了一个全长cDNA克隆，该克隆对应于来自小麦的转录本衍生片段（TDF108），据报道该克隆在含有针对镰刀菌传播抗性的数量性状基因座Qfhs.ndsu-3BS的小麦基因型中特异性表达（Steiner等，2009） 。只能克隆与TF108有一些序列差异的cDNA。但是，表达此小麦UGT cDNA的毒素敏感酵母菌株未显示抗性表型。通过酵母中的异源表达产生全长cDNA以进行功能验证的主要困难是植物中存在大量的UGT超家族成员，在拟南芥中具有107个UGT基因加一些假基因，在草中具有约150个假定的UGT基因。我们得出结论，序列相似性和可诱导性都不是底物特异性的良好预测指标。

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