Identification and characterization of phytic acid synthesis mutations in oilseed rape

植酸在植物中起着重要的磷酸盐储存化合物的作用。虽然许多植酸合成和转运蛋白基因已在模式物种以及作物物种中被鉴定，但油菜籽中的相应基因、它们的表达和功能在很大程度上是未知的。本研究旨在鉴定植酸合成相关基因，并分离突变体以研究基因功能。此外，这些突变体可能对油菜育种有益。首先，我们将对油菜基因组进行比较序列分析，以确定所有植酸合成基因，然后测试它们在叶片和种子中的表达。我们将重点研究种子中植酸的积累，由于富含蛋白质的脱脂菜籽粕中的植酸含量高，限制了其在动物和人类营养中的应用。在对基因进行计算机鉴定后，我们将使用TILLING-by-Sequencing方法在EMS诱变的冬油菜群体中寻找植酸合成和转运蛋白基因内的错义和无义突变。单突变体将被表征并彼此杂交以产生低植酸双突变体。在第二个实验中，将通过CRISPR/Cas9技术诱导植酸合成基因内的突变，对选定的植酸突变体进行进一步表征，测量基因表达、酶或转运蛋白活性、发育中种子中的植酸积累以及肌醇磷酸中间体、可溶性磷酸盐和总磷酸盐，用于磷酸盐平衡，研究代谢通量。油菜植酸合成基因在E.大肠杆菌中的表达将证实候选基因的功能。

Phytic acid plays a major role as a phosphate storage compound in plants. While many phytic acid synthesis and transporter genes have been identified in model species as well as in crop species, the corresponding genes from rapeseed, their expression and function are largely unknown. This project aims at the identification of all genes involved in phytic acid synthesis and the isolation of mutants to study gene function. Moreover, those mutants could be beneficial for oilseed rape breeding First, we will perform a comparative sequence analysis of the rapeseed genome to identify all phytic acid synthesis genes that will then be tested for their expression in leaves and seeds.We will focus our studies on phytic acid accumulation in seeds, as high contents of phytic acid in the protein-rich defatted rapeseed meal limit its use in animal and human nutrition.After in silico identification of the genes, we will search an EMS mutagenized winter rapeseed population for missense and nonsense mutations within phytic acid synthesis and transporter genes using a TILLING-by-Sequencing approach. Single mutants will be characterized and crossed among each other to produce low phytic acid double mutants. In a second experiment, mutations within phytic acid synthesis genes will be induced by the CRISPR/Cas9 technique.Selected phytic acid mutants will be further characterized measuring gene expression, enzyme or transporter activity and phytic acid accumulation in developing seeds as well as inositol phosphate intermediates, soluble and total phosphate for phosphate balancing to study metabolic fluxes. Enzyme kinetics measurements after overexpression of rapeseed phytic acid synthesis genes in E. coli will confirm the function of the candidate genes.