通过引入靶向核酸酶（ZFN、TALEN）造成目标位点DNA双链断裂，在细胞内源修复系统作用下，可实现目标基因组精确定位修饰，是下一代生物遗传修饰技术的关键所在。本研究以靶向核酸酶技术体系在小麦中的构建为出发点，针对小麦基因组结构特性及ZFN、TALEN设计原理，着力搭建高效率、高活性、高特异性的小麦靶向核酸酶技术平台，重点提高ZFN、TALEN对小麦多倍体基因组同源序列的识别特异性及构建高效率筛选方案。在此基础上，针对小麦基因工程常用选择及标记基因、小麦1RS-1BL易位系黑麦碱基因，构建及筛选高效ZFN、TALEN，实现：（1）选择及标记基因的高效敲除；（2）黑麦碱基因的特异性敲除或基因簇缺失；（3）黑麦碱位点的BAR基因重组置换。进而结合常规育种途径，分离、筛选、固定各类型基因组修饰后代株系，为合理、有效应用靶向核酸酶进行小麦基因功能分析及种质创新提供理论依据和物质基础。

Engineered nucleases, such as Zinc Finger nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN), have been recently utilized to create high efficient targeted genome modification in model plants and animals. Such nucleases can be designed to recognize and induce double-strand DNA breaks at specific genomic loci. These breaks are subjected to cellular DNA repair processed that often result in sequence alteration at target loci with remarkably high frequencies. Although nuclease-mediated genome modification has been demonstrated in several plant species, including Arabidopsis, tobacco and maize, it has been indicated that the technology platform has to be customized for each target species based on its genome complexity, polyploidy and suitable transformation tools. In this proposal, we aim to first establish a high efficient ZFN and TALEN platform for precise genome modification in wheat. ZFNs and TALENs with high efficacy and sequence specificity will be designed, evaluated and tested taking into account of complexity and polyploidy of wheat genome. Customized ZFNs or TALENs will be made to target exogenous selection or marker genes and endogenous secalin gene. Specific aims include: 1) to demonstrate highly efficient gene disruption in pre-integrated selection marker genes; 2) to generate mutant lines with disrupted secalin gene or gene family; 3) to demonstrate and optimize targeted gene replacement at the secalin locus. Modified wheat lines will be further followed through traditional breeding for phenotype test in progenies. Completion of the proposed research will establish a new paradigm in wheat genetic manipulation for basic and applied research.