RNA interference and low-allergenic plant-derived food under changing environmental conditions

番茄（Solanum esculentum）可缓解致敏患者的过敏症状，如口腔过敏综合征、接触性荨麻疹综合征或过敏性休克。到目前为止，至少有13种蛋白质与番茄中的IgE结合有关。这些IgE结合蛋白中的四种已被归类为过敏原。在过去的几年里，我们已经建立了番茄果实作为模型，以测试分子策略，以产生低过敏性食品。该子项目的主要目的是（i）提供RNAi技术可用于生产低过敏性食品的原理证明，即使需要靶向多种过敏原，并且如果涉及具有基本细胞功能的过敏原，（ii）探索靶向基因组修饰的新技术和（iii）通过随机诱变结合酵母中的功能选择来研究profilin的过敏性结构。为了实现我们的第一个目标，我们已经开发了一种育种方案，以联合收割机下调番茄中多达8种IgE结合蛋白。第二个目标将通过使用定制的TALE核酸酶来诱变Lyc e 3来实现。为了以随机的方式识别profilins的过敏原决定因素，我们开发了一种基于酵母的筛选平台，以允许选择功能性和低过敏性的profilin变体。该系统已成功地用于鉴定低变应原性Lyc e 1，并将应用于筛选来自其他植物来源的低变应原性profilin变体，如Bet v 2或API g 4。在反向实验中，我们将诱变酵母profilin以测试它是否可能将非过敏性蛋白质转化为过敏原。

Tomato (Solanum esculentum) elicits allergic symptoms such as oral allergy syndrome, contact urticarial syndrome or anaphylactic shock in sensitized patients. So far at least 13 proteins have been associated with IgE-binding in tomato. Four of these IgE-binding proteins have been classified as allergens. Over the last years we have established tomato fruits as models to test molecular strategies to generate hypoallergenic food. Major aims of this sub-project are (i) to provide proof of principle that RNAi technologies can be used to generate hypoallergenic food even if multiple allergens need to be targeted and if allergens with essential cellular functions are involved, (ii) to explore new technologies for targeted genome modifications and (iii) to investigate allergenic structures of profilin by random mutagenesis combined with functional selection in yeast. To achieve our first aim we have developed a breeding scheme to combine down-regulation of up to eight IgE-binding proteins from tomato. The second goal will be reached by using customized TALE nucleases for mutagenesis of Lyc e 3. To identify allergenic determinants of profilins in a random fashion we have developed a yeast-based screening platform to allow selection for functional and hypoallergenic profilin variants. This system has successfully been used to identify hypoallergenic Lyc e 1 and will be applied to screen for hypoallergenic profilin variants from other plant sources such as Bet v 2 or Api g 4. In a reverse experiment we will mutagenize yeast profilin to test whether it is possibly to convert a non-allergenic protein into an allergen.